SMART MODE:

NORMAL GENOMIC

• Kubiak RJ, Yue X, Hondal RJ, Mihai C, Tsai MD, Bruzik KS
• Involvement of the Arg-Asp-His catalytic triad in enzymatic cleavage of the phosphodiester bond.
• Biochemistry. 2001; 40: 5422-32
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• Phosphatidylinositol-specific phospholipase C (PI-PLC) catalyzes the cleavage of the P-O bond in phosphatidylinositol via intramolecular nucleophilic attack of the 2-hydroxyl group of inositol on the phosphorus atom. Our earlier stereochemical and site-directed mutagenesis studies indicated that this reaction proceeds by a mechanism similar to that of RNase A, and that the catalytic site of PI-PLC consists of three major components analogous to those observed in RNase A, the His32 general base, the His82 general acid, and Arg69 acting as a phosphate-activating residue. In addition, His32 is associated with Asp274 in forming a catalytic triad with inositol 2-hydroxyl, and His82 is associated with Asp33 in forming a catalytic diad. The focus of this work is to provide a global view of the mechanism, assess cooperation between various catalytic residues, and determine the origin of enzyme activation by the hydrophobic leaving group. To this end, we have investigated kinetic properties of Arg69, Asp33, and His82 mutants with phosphorothioate substrate analogues which feature leaving groups of varying hydrophobicity and pK(a). Our results indicate that interaction of the nonbridging pro-S oxygen atom of the phosphate group with Arg69 is strongly affected by Asp33, and to a smaller extent by His82. This result in conjunction with those obtained earlier can be rationalized in terms of a novel, dual-function triad comprised of Arg69, Asp33, and His82 residues. The function of this triad is to both activate the phosphate group toward the nucleophilic attack and to protonate the leaving group. In addition, Asp33 and His82 mutants displayed much smaller degrees of activation by the fatty acid-containing leaving group as compared to the wild-type (WT) enzyme, and the level of activation was significantly reduced for substrates featuring the leaving group with low pK(a) values. These results strongly suggest that the assembly of the above three residues into the fully catalytically competent triad is controlled by the hydrophobic interactions of the enzyme with the substrate leaving group.

• Zaikova TO, Rukavishnikov AV, Birrell GB, Griffith OH, Keana JF
• Synthesis of fluorogenic substrates for continuous assay of phosphatidylinositol-specific phospholipase C.
• Bioconjug Chem. 2001; 12: 307-13
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• An improved synthesis of fluorogenic substrate analogues for phosphatidylinositol-specific phospholipase C (PI-PLC) is described. The water-soluble substrates, which are derived from fluorescein, are not fluorescent until cleaved by the enzyme, and provide a convenient means to continuously monitor PI-PLC activity. The improvement in the synthesis lies in the method used to protect the hydroxyl groups of the inositol portion of the substrate molecule and allows a milder deprotection procedure to be used. The result is a much more reproducible synthesis of the substrate. The improved procedure has been employed to synthesize a series of fluorogenic substrates, which differ in the length of the aliphatic tail attached to the fluorescein portion of the molecule. The length of the tail was found to have a significant effect on the rate of cleavage of these substrates.

• Otterhag L, Sommarin M, Pical C
• N-terminal EF-hand-like domain is required for phosphoinositide-specific phospholipase C activity in Arabidopsis thaliana.
• FEBS Lett. 2001; 497: 165-70
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• Phosphoinositide-specific phospholipase C's (PI-PLCs) are ubiquitous in eukaryotes, from plants to animals, and catalyze the hydrolysis of phosphatidylinositol 4,5-bisphosphate into the two second messengers inositol 1,4,5-trisphosphate and diacylglycerol. In animals, four distinct subfamilies of PI-PLCs have been identified, and the three-dimensional structure of one rat isozyme, PLC-delta1, determined. Plants appear to contain only one gene family encoding PI-PLCs. The catalytic properties of plant PI-PLCs are very similar to those of animal enzymes. However, very little is known about the regulation of plant PI-PLCs. All plant PI-PLCs comprise three domains, X, Y and C2, which are also conserved in isoforms from animals and yeast. We here show that one PI-PLC isozyme from Arabidopsis thaliana, AtPLC2, is predominantly localized in the plasma membrane, and that the conserved N-terminal domain may represent an EF-hand domain that is required for catalytic activity but not for lipid binding.

• Fritsky IO, Ott R, Pritzkow H, Kramer R
• An allosteric synthetic catalyst: metal ions tune the activity of an artificial phosphodiesterase.
• Chemistry. 2001; 7: 1221-31
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• A trinuclear metal complex of general formula (L-H)M3(Mf)2 represents the first allosteric low molecular weight catalyst. L is a polyaza ligand having a tetradentate and two bidentate metal binding sites, Ms is a "structural" (allosteric) metal, and Mf are functional (catalytic) metals which interact with a substrate. In mononuclear [(L-H)Ms]+ complexes [(L-H)Cu(MeOH)]ClO4 (1a). [(L-H)Cu]NO3 x 2H2O (1b), [(L-H)Ni]ClO4 x 4H2O (2), and [(L-H)Pd]ClO4 x 2H2O (3), prepared from L and M2+ salts, the metal is strongly bound by an in-plane N4-coordination (confirmed by X-ray crystal structure determination of la). Formation of trinuclear complexes [(L-H)MsCu2]5+, with two functional Cu2+ ions coordinated to the bidentate sites of L, was evidenced in solution by photometric titration and by isolation of [(L-H)Cu3][PO4][ClO4]2 x 9H2O (4). The trinuclear complexes catalyze the cleavage of RNA-analogue 2-(hydroxypropyl)-p-nitrophenyl phosphate (HPNP), an activated phosphodiester. From a kinetic analysis of the cleavage rate at various HPNP concentrations, parameters KHPNP (the equilibrium constant for binding of HPNP to [(L-H)MsCu2]5+ and kcat (first-order rate constant for cleavage of HPNP when bound to the catalyst) were derived: KM= 170 (Ms= Cu2+), 340 (Ms = Ni2+), 2,600 (Ms = Pd2+) M(-1), kcat = 17 x 10(-3) (Ms= Cu2+) 3.1 x 10(-3) (Ms=Ni2+), 0.22 x 10(-3) (Ms = Pd2+) s(-1). Obviously, the nature of the allosteric metal ion Ms strongly influences both substrate affinity and reactivity of the catalyst [(L-H)MsCu2]5+. Our interpretation of this observation is that subtle differences in the ionic radius of Ms and in its tendency to distort the N4-Ms coordination plane have a significant influence on the conformation of the catalyst (i.e., preorganization of functional Cu2+ ions) and thus on catalytic activity.

• Kravchuk AV, Zhao L, Kubiak RJ, Bruzik KS, Tsai MD
• Mechanism of phosphatidylinositol-specific phospholipase C: origin of unusually high nonbridging thio effects.
• Biochemistry. 2001; 40: 5433-9
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• Phosphatidylinositol-specific phospholipase C (PI-PLC) has been proposed previously to employ a catalytic mechanism highly reminiscent of that of ribonuclease A (RNase A). Both catalytic sites are comprised of two histidine side chains acting as a general base-general acid pair and a phosphate-activating residue: an arginine in the case of PI-PLC and a lysine in RNase A. Despite these structural similarities, the PI-PLC reaction is slowed 10(5)-fold upon substitution of one of the phosphate nonbridging oxygen atoms with sulfur, whereas a much smaller effect is observed in the analogous RNase A reaction. Here, we report a systematic study of this property in PI-PLC, conducted by means of site-directed chemical modification of a cysteine residue replacing the arginine at position 69. The results show that mutant enzymes featuring bidentate side chains at this position display significantly higher activity, higher thio effects, and greater stereoselectivity than do those with monodentate side chains. The results suggest that the bidentate nature of Arg69 is the origin of the large thio effects and stereoselectivity in PI-PLC. We propose that in addition to binding the phosphate, the function of arginine 69 is to bring the phosphate group and the 2-OH group of inositol into proximity and to induce proper alignment for nucleophilic attack, and possibly to lower the pK(a) of the 2-OH. The results presented here could be important to mechanisms of phosphoryl transfer enzymes in general, suggesting that a major part of thio effects observed in enzymatic phosphoryl transfer reactions can originate from factors other than direct interaction between a side chain and a phosphate group, and caution the use of the absolute magnitude of the thio effect as an indicator of the strength of such interactions.

• Cocco L, Martelli AM, Mazzotti G, Barnabei O, Manzoli FA
• Inositides and the nucleus: phospholipase Cbeta family localization and signaling activity.
• Adv Enzyme Regul. 2000; 40: 83-95

• Cocco L, Rhee SG, Gilmour RS, Manzoli FA
• Inositide-specific phospholipase C signalling in the nucleus.
• Eur J Histochem. 2000; 44: 45-50

• Dwivedi Y, Rizavi HS, Rao JS, Pandey GN
• Modifications in the phosphoinositide signaling pathway by adrenal glucocorticoids in rat brain: focus on phosphoinositide-specific phospholipase C and inositol 1,4,5-trisphosphate.
• J Pharmacol Exp Ther. 2000; 295: 244-54
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• The hypothalamic-pituitary-adrenal (HPA) axis has been shown to be involved in mood and behavior. The possibility that adrenal glucocorticoids regulate components of the phosphatidylinositol (PI) signal transduction pathway was investigated. Two different doses of corticosterone (CORT) pellets (50 or 100 mg) were implanted in normal and bilaterally adrenalectomized (ADX) rats, and CORT regulation of the expression of G(q) alpha protein, phospholipase C (PLC) isozymes, inositol 1,4,5-trisphosphate receptor (IP(3)R) isoforms, and of PI-PLC activity, [(3)H]IP(3) binding to IP(3)Rs, and IP(3) levels were measured in various brain areas after 1 or 14 days. Fourteen days of CORT pellet implantation into normal rats dose dependently decreased PI-PLC activity and selectively the mRNA and protein expression of PLC beta(1) isozyme in cortex and hippocampus. Bilateral ADX caused the opposite changes in these measures, and simultaneous CORT pellet implantation into ADX rats reversed these effects. Furthermore, 14 days of CORT treatment of normal rats increased [(3)H]IP(3) binding to IP(3)Rs and decreased IP(3) levels in cortex, hippocampus, and cerebellum, without any changes in expression of IP(3)R-I, IP(3)R-II, or IP(3)R-III isoform. On the other hand, ADX decreased [(3)H]IP(3) binding and increased levels of IP(3), and simultaneous CORT treatment of ADX rats prevented these changes. ADX or CORT treatment had no significant effects on the expression of G(q/11) alpha protein. These results suggest that manipulation of the HPA axis alters various components of the PI signaling pathway in rat brain, which may have physiological relevance to the HPA axis-mediated changes in mood and behavior.

• Kanematsu T, Yoshimura K, Hidaka K, Takeuchi H, Katan M, Hirata M
• Domain organization of p130, PLC-related catalytically inactive protein, and structural basis for the lack of enzyme activity.
• Eur J Biochem. 2000; 267: 2731-7
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• The 130-kDa protein (p130) was isolated as a novel inositol 1,4, 5-trisphosphate [Ins(1,4,5)P3]-binding protein similar to phospholipase C-delta1 (PLC-delta1), but lacking catalytic activity [Kanematsu, T., Takeya, H., Watanabe, Y., Ozaki, S., Yoshida, M., Koga, T., Iwanaga, S. & Hirata, M. (1992) J. Biol. Chem. 267, 6518-6525; Kanematsu, T., Misumi, Y., Watanabe, Y., Ozaki, S., Koga, T., Iwanaga, S., Ikehara, Y. & Hirata, M. (1996) Biochem. J. 313, 319-325]. To test experimentally the domain organization of p130 and structural basis for lack of PLC activity, we subjected p130 to limited proteolysis and also constructed a number of chimeras with PLC-delta1. Trypsin treatment of p130 produced four major polypeptides with molecular masses of 86 kDa, 55 kDa, 33 kDa and 25 kDa. Two polypeptides of 86 kDa and 55 kDa started at Lys93 and were calculated to end at Arg851 and Arg568, respectively. Using the same approach, it has been found that the polypeptides of 33 kDa and 25 kDa are likely to correspond to regions between Val569 and Arg851 and Lys869 and Leu1096, respectively. All the proteolytic sites were in interconnecting regions between the predicted domains, therefore supporting domain organization based on sequence similarity to PLC-delta1 and demonstrating that all domains of p130, including the unique region at the C-terminus, are stable, tightly folded structures. p130 truncated at either or both the N-terminus (94 amino acids) and C-terminus (851-1096 amino acids) expressed in COS-1 cells showed no catalytic activity, indicating that p130 has intrinsically no PLC activity. A number of chimeric molecules between p130 and PLC-delta1 were constructed and assayed for PLC activity. It was shown that structural differences in interdomain interactions exist between the two proteins, as only some domains of p130 could replace the corresponding structures in PLC-delta1 to form a functional enzyme. These results suggest that p130 and the related proteins could represent a new protein family that may play some distinct role in cells due to the capability of binding Ins(1,4,5)P3 but the lack of catalytic activity.

• Kim MJ, Kim E, Ryu SH, Suh PG
• The mechanism of phospholipase C-gamma1 regulation.
• Exp Mol Med. 2000; 32: 101-9
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• Phospholipase C (PLC)1 hydrolyzes phosphatidylinositol 4,5-bisphosphate to generate the second messengers, inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). IP3 induces a transient increase in intracellular free Ca2+, while DAG directly activates protein kinase C. Upon stimulation of cells with growth factors, PLC-gamma1 is activated upon their association with and phosphorylation by receptor and non-receptor tyrosine kinases. In this review, we will focus on the activation mechanism and regulatory function of PLC-gamma1.

• Di Pietro R, Miscia S, Cataldi A, Rana R
• Age-dependent variations in the expression of PLC isoforms upon mitogenic stimulation of peripheral blood T cells from healthy donors.
• Br J Haematol. 2000; 111: 1209-14
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• The activation of phosphoinositide-specific phospholipase C (PLC) is one of the early responses to various growth factors and it is known that these functions are altered with ageing. In the present investigation, the expression and cellular distribution of PLC isoenzymes in immunocompetent T lymphocytes from a test group of healthy individuals over 65 years old and a comparison group of healthy donors below 30 years old were compared using Western blot and confocal microscopy. All lymphocyte samples responded biochemically to phytohaemagglutinin (PHA) stimulation, as shown by at least 10-fold increases in the beta(3) isoform and nuclear translocation of the beta(1), beta(2), beta(3), beta(4), gamma(1) and gamma(2) isoforms; however, consistent differences in the expression of the beta(2) isoform in unstimulated T cells and of the beta(2) and gamma(2) isoforms in stimulated T cells suggest that altered functions of the PLC pathway may have an age-dependent impact on signal transduction events.

• Jepson M, Titball R
• Structure and function of clostridial phospholipases C.
• Microbes Infect. 2000; 2: 1277-84
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• A range of clostridial species produce phospholipases C. The zinc metallo phospholipases C have related sequences but different properties. All of these enzymes may be arranged, like alpha-toxin as two-domain proteins. Differences in enzymatic, haemolytic and toxic properties may be explained by differences in amino acids at key positions.

• Hess JA, Buchanan FG, Ryder S, Exton JH
• Altered activation of phospholipase D by lysophosphatidic acid and endothelin-1 in mouse embryo fibroblasts lacking phospholipase C-gamma1.
• Cell Signal. 2000; 12: 37-45
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• Lysophosphatidic acid (LPA) and endothelin-1 (ET-1) activate phospholipase D (PLD) in many cell types. To see if phospholipase C-gamma1 plays a role, we used embryonic fibroblasts from mice in which the PLCgamma1 gene was disrupted. Surprisingly, the effect of LPA on inositol phosphate accumulation was increased in these PLCgamma1-/- cells, whereas that of ET-1 was completely abrogated. When PLD activity was measured, the response to LPA was also enhanced and the response to ET-1 lost in the PLCgamma1-/- cells. Treatment of these cells with ionomycin and oleoyl acetyl glycerol to mimic PLC stimulation restored PLD activity. Treatment of either PLCgamma1+/+ and PLCgamma1-/- cells with tyrosine kinase inhibitors did not inhibit LPA- or ET-1-induced PLD activity. Moreover, LPA and ET-1 treatment of PLCgamma1+/+ and PLCgamma1-/- cells did not cause tyrosine phosphorylation of PLC-gamma1 or PLC-gamma2. In summary, these results show that the altered PLD responses to LPA and ET-1 in PLCgamma1-/- are due to changes in PLC activity and do not involve tyrosine kinase activity.

• Jones GA, Wu Y
• Effect of limited proteolysis on phospholipase C-gamma1 kinetics.
• Arch Biochem Biophys. 2000; 375: 229-39
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• Phospholipase C-gamma1 is a tightly regulated, multidomain protein that generates the second messengers inositol 1,4,5-trisphosphate and diacylglycerol. Kinetic analysis reveals that phospholipase C-gamma1 displays apparent allosteric behavior. A previous study determined that proteolytic cleavage of the SH domain region of phospholipase C-gamma1 yields an activated form of the enzyme (A. W. Fernald, G. A. Jones, and G. Carpenter Biochem. J. 302, 508, 1994). In this study, we show that activation of phospholipase C-gamma1 by proteolysis decreases both the cooperativity and the half-maximal value of the enzyme for substrate. Kinetic analysis revealed that the mole fraction of phosphatidylinositol 4,5-bisphosphate (PIP(2)) that resulted in half-maximal PIP(2) hydrolysis (S(0.5)) was lower for proteolyzed than uncleaved phospholipase C-gamma1 (0.08 mole fraction vs 0.18 mole fraction of PIP(2)). The cooperativity index was lower for proteolyzed than full-length phospholipase C-gamma1 (n = 2.5 vs n = 4). Kinetic analysis also revealed that the estimated dissociation constant was lower for phospholipase C-gamma1 that had been subjected to proteolysis (0.1 mM vs 1.0 mM PIP(2) for cleaved vs uncleaved phospholipase C-gamma1, respectively). It was previously hypothesized that activation of phospholipase C-gamma1 requires a conformational change that results in increased accessibility of substrate to the active site and that the SH domain of the enzyme is involved in the activation event. These experiments support the hypothesis that a portion of the protein covers the active site, allosterically inhibiting the enzyme, and that the removal of this "lid" domain activates the enzyme.

• Furuya T, Kashuba C, Docampo R, Moreno SN
• A novel phosphatidylinositol-phospholipase C of Trypanosoma cruzi that is lipid modified and activated during trypomastigote to amastigote differentiation.
• J Biol Chem. 2000; 275: 6428-38
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• The phosphoinositide (PI)-specific phospholipase C gene (TcPI-PLC) of the protozoan parasite Trypanosoma cruzi was cloned, sequenced, expressed in Escherichia coli, and the protein product (TcPI-PLC) was shown to have enzymatic characteristics similar to those of mammalian delta-type PI-PLCs. The TcPI-PLC gene is expressed at high levels in the epimastigote and amastigote stages of the parasite, and its expression is induced during the differentiation of trypomastigotes into amastigotes, where TcPI-PLC associates with the plasma membrane and increases its catalytic activity. In contrast to other PI-PLCs described so far, the deduced amino acid sequence of TcPI-PLC revealed some unique features such as an N-myristoylation consensus sequence at its amino-terminal end, lack of an apparent pleckstrin homology domain and a highly charged linker region between the catalytic X and Y domains. TcPI-PLC is lipid modified in vivo, as demonstrated by metabolic labeling with [(3)H]myristate and [(3)H]palmitate and fatty acid analysis of the immunoprecipitated protein, and may constitute the first example of a new group of PI-PLCs.

• Goni FM, Alonso A
• Membrane fusion induced by phospholipase C and sphingomyelinases.
• Biosci Rep. 2000; 20: 443-63
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• In the past decade lipid vesicle fusion induced by either bacterial PC-preferring phospholipase C, phosphatidylinositol-specific phospholipase C, sphingomyelinase, or a combination of phospholipase C and sphingomyelinase has been demonstrated. In the present paper, the experimental evidence is reviewed, and discussed in terms of the underlying molecular mechanisms of fusion, and of the possible physiological relevance of these findings.

• Rebecchi MJ, Pentyala SN
• Structure, function, and control of phosphoinositide-specific phospholipase C.
• Physiol Rev. 2000; 80: 1291-335
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• Phosphoinositide-specific phospholipase C (PLC) subtypes beta, gamma, and delta comprise a related group of multidomain phosphodiesterases that cleave the polar head groups from inositol lipids. Activated by all classes of cell surface receptor, these enzymes generate the ubiquitous second messengers inositol 1,4, 5-trisphosphate and diacylglycerol. The last 5 years have seen remarkable advances in our understanding of the molecular and biological facets of PLCs. New insights into their multidomain arrangement and catalytic mechanism have been gained from crystallographic studies of PLC-delta(1), while new modes of controlling PLC activity have been uncovered in cellular studies. Most notable is the realization that PLC-beta, -gamma, and -delta isoforms act in concert, each contributing to a specific aspect of the cellular response. Clues to their true biological roles were also obtained. Long assumed to function broadly in calcium-regulated processes, genetic studies in yeast, slime molds, plants, flies, and mammals point to specific and conditional roles for each PLC isoform in cell signaling and development. In this review we consider each subtype of PLC in organisms ranging from yeast to mammals and discuss their molecular regulation and biological function.

• Jones GA, Lazarchic M
• Phosphatidylinositol-specific phospholipase C-gamma1 undergoes pH-induced activation and conformational change.
• Biochim Biophys Acta. 2000; 1487: 209-21
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• Phospholipase C-gamma1 displayed sigmoidal kinetics with a S(0.5) value of 0.17 mole fraction PIP(2) when assayed at pH 6.8 using detergent:lipid mixed micelles. The pH optimum for hydrolysis of phosphatidylinositol 4,5-bisphosphate by phospholipase C-gamma1 was dependent on the mole fraction of substrate in the micelle. The pH optimum was 5.5 when the enzyme was assayed below the S(0.5). The pH optima shifted to a pH range of 6.0-6.3 when the enzyme was assayed above the S(0.5). The kinetic parameters for phospholipase C-gamma1 assayed at various pH values from pH 7.0 to 5.0 yielded similar n values (n=4), but the constant, K', decreased from 1x10(-2) (mole fraction)(2) at pH 7.0 to 1x10(-5) (mole fraction)(2) at pH 5.0. Maximum enzyme specificity occurred at pH values below pH 6.0 as determined by the plot of logk(cat)/S(0.5) versus pH. Intrinsic fluorescence spectroscopy revealed that at a pH value above 7.0 or below 6.3, tryptophan quenching occurred. Fluorescence quenching experiments performed with acrylamide determined phospholipase C-gamma1 incubated at pH 5.0 had a larger collisional quenching constant than enzyme incubated at pH 7.0. Lowering the pH to 5.0 apparently resulted in interior tryptophans becoming more solvent accessible. These data suggest that pH may activate phospholipase C-gamma1 by disrupting ionizable groups leading to a conformational change.

• Peruzzi D et al.
• Identification and chromosomal localisation by fluorescence in situ hybridisation of human gene of phosphoinositide-specific phospholipase C beta(1).
• Biochim Biophys Acta. 2000; 1484: 175-82
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• Members of phosphoinositide-specific phospholipase C (PLC) families are central intermediary in signal transduction in response to the occupancy of receptors by many growth factors. Among PLC isoforms, the type beta(1) is of particular interest because of its reported nuclear localisation in addition to its presence at the plasma membrane. It has been previously shown that both the stimulation and the inhibition of the nuclear PLCbeta(1) under different stimuli implicate PLCbeta(1) as an important enzyme for mitogen-activated cell growth as well as for murine erythroleukaemia cell differentiation. The above findings hinting at a direct involvement of PLCbeta(1) in controlling the cell cycle in rodent cells, and the previously reported mapping of its gene in rat chromosome band 3q35-36, a region frequently rearranged in rat tumours induced by chemical carcinogenesis, prompted us to identify its human homologue. By screening a human foetal brain cDNA library with the rat PLCbeta(1) cDNA probe, we have identified a clone homologous to a sequence in gene bank called KIAA 0581, which encodes a large part of the human PLCbeta(1). By using this human cDNA in fluorescence in situ hybridisation on human metaphases, it has been possible to map human PLCbeta(1) on chromosome 20p12, confirming the synteny between rat chromosome 3 and human chromosome 20 and providing a novel locus of homology between bands q35-36 in rat and p12 in man. Since band 20p12 has been recently reported amplified and/or deleted in several solid tumours, the identification and chromosome mapping of human PLCbeta(1) could pave the way for further investigations on the role exerted both in normal human cells and in human tumours by PLCbeta(1), which has been shown to behave as a key signalling intermediate in the control of the cell cycle.

• Fujii M, Ohtsubo M, Ogawa T, Kamata H, Hirata H, Yagisawa H
• Real-time visualization of PH domain-dependent translocation of phospholipase C-delta1 in renal epithelial cells (MDCK): response to hypo-osmotic stress.
• Biochem Biophys Res Commun. 1999; 254: 284-91
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• Green fluorescent protein (GFP)-tagged phospholipase C (PLC)-delta1 and its mutants were expressed in Madin-Darby canine kidney (MDCK) cells. GFP-PLC-delta1 or the GFP-tagged pleckstrin homology (PH) domain of PLC-delta1 itself was found to be predominantly localized at the plasma membrane. The DeltaPH mutant or a site-directed mutant containing a PH domain which does not bind inositol 1,4, 5-trisphosphate and cannot hydrolyze phosphatidylinositol 4, 5-bisphosphate in vitro was seen only in the cytosol. In living MDCK cells hypo-osmotic stress caused a rapid dissociation of GFP-PLC-delta1 from the plasma membrane, which coincided with phosphoinositide breakdown. A PLC inhibitor, U73122, blocked this translocation, but depletion of extracellular Ca2+ had no effect. The translocation was reversed by replacement with an iso-osmotic buffer. Our results demonstrate that the PH domain plays a critical role in the membrane targeting of PLC-delta1 and that the intracellular distribution of the enzyme is regulated by osmotic stress-driven phosphoinositide turnover.

• Hendrickson HS, Hendrickson EK
• Binding of phosphatidylinositol-specific phospholipase C to phospholipid interfaces, determined by fluorescence resonance energy transfer.
• Biochim Biophys Acta. 1999; 1440: 107-17
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• Dissociation constants for binding of phosphatidylinositol-specific phospholipase C from Bacillus cereus (bcPI-PLC) and the mammalian phosphatidylinositol-specific phospholipase C-delta(1) to lipid interfaces containing phosphoinositol, phosphocholine, and phosphomethanol head groups were determined by fluorescence resonance energy transfer. Dansyl-labeled lipid probes were used as acceptors, with intrinsic tryptophan of the enzyme as the donor. Titration of protein into lipid provided data from which K(d) and N, the limiting number of lipid molecules per protein bound, were calculated by non-linear regression analysis of exact binding equations. These results were compared with apparent K(m) values from kinetic data. K(d) values in the low microM range in terms of lipid monomers or low nM range in terms of binding sites were calculated with good fits of experimental data to theoretical binding curves. bcPI-PLC binds with high affinity to PI interfaces, slightly lower affinity to PC interfaces, and much lower affinity to PM interfaces. The mammalian enzyme also binds with high affinity to PI interfaces, but shows little or no binding with PC interfaces under similar concentration conditions. These K(d) values correlate reasonably with apparent K(m) values from kinetic experiments.

• Frohman MA, Morris AJ
• Phospholipase D structure and regulation.
• Chem Phys Lipids. 1999; 98: 127-40
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• The recent identification of cDNA clones for phospholipase D has opened the door to new types of investigations into its structure and regulation. PLD activity has been found to be encoded by at least two different genes that contain catalytic domains that relate their mechanism of action to phosphodiesterases. In vivo roles for PLD suggest that it may be important for multiples steps in regulated secretion and membrane biogenesis.

• Rukavishnikov AV, Zaikova TO, Birrell GB, Keana JF, Griffith OH
• Synthesis of a new fluorogenic substrate for the continuous assay of mammalian phosphoinositide-specific phospholipase C.
• Bioorg Med Chem Lett. 1999; 9: 1133-6
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• The synthesis of a fluorogenic substrate for mammalian phosphoinositide-specific phospholipase C is described. The substrate, based on the widely used fluorescein molecule, is a water-soluble substrate analog of phosphatidylinositol-4-phosphate. The fluorogenic substrate 2 is shown to be a sensitive substrate for human PI-PLC-delta1 in a continuous assay.

• Banno Y, Nozawa Y
• [Phospholipase C]
• Tanpakushitsu Kakusan Koso. 1999; 44: 989-97

• Rigual R, Cachero MT, Rocher A, Gonzalez C
• Hypoxia inhibits the synthesis of phosphoinositides in the rabbit carotid body.
• Pflugers Arch. 1999; 437: 839-45
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• Hypoxic transduction in the carotid body (CB) is regulated by several systems of second messengers, but the role of the phospholipase C system has not been studied. The aim of the present study was to characterize the turnover rate of inositol phosphates (InsPs) and phosphoinositides (PIs) and their modifications by hypoxia in the rabbit CB in vitro. In CBs, in which the PIs had been labelled previously with 3H-myo-inositol, hypoxia in the presence of LiCl did not modify the accumulation of 3H-InsPs, whilst exposure to hypoxia during the loading period in the presence of LiCl reduced the accumulation of 3H-InsPs by more than 50%. Endogenous levels of inositol 1,4,5-trisphosphate were unaltered by hypoxia. Synthesis of 3H-PIs from 3H-myo-inositol was markedly inhibited by hypoxia in the CB, but not in the rat superior cervical ganglion used as control tissue. Levels of 3H-phosphatidylinositol (3H-PtdIns), 3H-phosphatidylinositol 4-monophosphate and 3H-phosphatidylinositol 4,5-bisphosphate were similarly decreased, indicating that inhibition occurs at a step prior to PtdIns synthesis. It is concluded that the phospholipase C system of second messengers does not play a significant role in the short-term regulation of hypoxic transduction cascade. It can be speculated that the decrease in PI availability produced by hypoxia might be involved in the functional changes observed in the CB on chronic hypoxic exposure.

• Pandey GN et al.
• Low phosphoinositide-specific phospholipase C activity and expression of phospholipase C beta1 protein in the prefrontal cortex of teenage suicide subjects.
• Am J Psychiatry. 1999; 156: 1895-901
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• OBJECTIVE: The enzyme phosphoinositide-specific phospholipase C (PI-PLC) is a component of the phosphoinositide signal transduction system. Other components of this system have been found to be abnormal in adults and adolescents who have committed suicide, and so the authors examined whether PI-PLC activity and protein expression of PLC isozymes are abnormal in postmortem brains of teenage suicide subjects. METHOD: PI-PLC activity and protein expression of the PLC beta1, delta1, and gamma1 isozymes were examined in Brodmann's areas 8 and 9 of postmortem brains obtained from 18 teenage suicide subjects and 18 matched comparison subjects. PI-PLC activity was determined by enzymatic assay, and protein expression of the PLC isozymes was determined by the Western blot technique. RESULTS: Compared with the normal subjects, the teenage suicide subjects had significantly lower PI-PLC activity and immunolabeling of the specific PLC beta1 isozyme in both membrane and cytosol fractions of Brodmann's areas 8 and 9 combined (prefrontal cortex). There was also a significant correlation between PI-PLC activity and protein levels of the PLC beta1 isozyme in the brains of the teenage suicide subjects. There was no significant difference in PI-PLC activity or level of PLC beta1 protein between the suicide subjects with a history of mental disorders and those with no history of mental disorders; however, both groups had significantly lower PI-PLC activity and expression of PLC beta1 protein than the normal subjects. CONCLUSIONS: Low PI-PLC activity and expressed levels of the PLC beta1 isozyme in postmortem brains of suicide subjects may have clinical relevance in the pathophysiology of suicidal behavior.

• Sekiya F, Bae YS, Rhee SG
• Regulation of phospholipase C isozymes: activation of phospholipase C-gamma in the absence of tyrosine-phosphorylation.
• Chem Phys Lipids. 1999; 98: 3-11
• Display abstract

• Activation of PLC-gamma isozymes in response to various agonists involves tyrosine phosphorylation of the effector enzymes. Recent evidence indicates that PLC-gamma isozymes are additionally activated by phosphatidic acid, phosphatidylinositol 3,4,5-trisphosphate and arachidonic acid in the absence of PLC-gamma tyrosine phosphorylation. These lipid-derived messengers are the immediate products of phospholipase D, phosphatidylinositol 3-kinase, and phospholipase A2, enzymes which are often stimulated along with PLC-gamma in response to an agonist. Furthermore, phosphatidylinositol 4,5-bisphosphate acts as a substrate for both PLC-gamma and phosphatidylinositol 3-kinase and as an activator for phospholipase D and phospholipase A2. These results reveal an elaborate mechanism of cross-talk and mutual regulation between four effector enzymes that participate in receptor signaling by acting on phospholipids.

• Waite M
• The PLD superfamily: insights into catalysis.
• Biochim Biophys Acta. 1999; 1439: 187-97
• Display abstract

• Knowledge of the PLD superfamily is rapidly expanding and new insights into the mechanism and regulation of the superfamily are rapidly emerging. The recent structural analysis and use of mutant proteins suggest a mechanism that involves two active sites acting in concert. While a number of residues are required for activity, it appears most likely that a histidine is the residue that becomes covalently linked to phosphatidate in catalysis. Evidence for these proposals is covered in this article.

• Han JS, Hyun BC, Kim JH, Shin I
• Fas-mediated activation of phospholipase D is coupled to the stimulation of phosphatidylcholine-specific phospholipase C in A20 cells.
• Arch Biochem Biophys. 1999; 367: 233-9
• Display abstract

• The activation of phospholipase D in murine B cell lymphoma A20 cells treated with anti-Fas monoclonal antibody has been investigated. Fas cross-linking resulted in a both dose- and time-dependent increases in phospholipase D activity. There was a nearly maximum saturated rise in phospholipase D activity at the dose of 200 ng/ml anti-Fas monoclonal antibody showing a fourfold increase within 3 h. Fas activation also caused an approximately twofold increase of phosphatidylcholine-specific phospholipase C activity and 1,2-diacylglycerol release, which could be blocked by 30 min pretreatment with the phosphatidylcholine-specific phospholipase C inhibitor D609 (50 microgram/ml). Pretreatment of D609 also effectively inhibited the translocation of protein kinase C betaI and betaII from the cytosol to the membrane and the activation of phospholipase D induced by Fas cross-linking, suggesting that 1, 2-diacylglycerol released from the cellular phosphatidylcholine pool through phosphatidylcholine-specific phospholipase C plays a major role in protein kinase C/phospholipase D activation. Anti-Fas monoclonal antibody failed to elicit phosphoinositide-specific phospholipase C activation and any changes in the intracellular Ca2+ level in A20 cells, indicating that the phosphoinositide-mediated pathway is not involved in this Fas signaling. Therefore, these results suggest that Fas-mediated phospholipase D activation may be a consequence of primary stimulation of phosphatidylcholine-specific phospholipase C and that phospholipase D may play a role in Fas cross-linking signaling downstream from phosphatidylcholine-specific phospholipase C.

• Otsuki M, Fukami K, Kohno T, Yokota J, Takenawa T
• Identification and characterization of a new phospholipase C-like protein, PLC-L(2).
• Biochem Biophys Res Commun. 1999; 266: 97-103
• Display abstract

• We have isolated a cDNA encoding a novel protein, PLC-L(2), with homology to the phospholipase C-like protein PLC-L and delta-type phospholipase C. PLC-L(2) contains a relatively well-conserved PH domain, PLC catalytic region, and X and Y domains. However, it did not have PLC activity. This inactivation was thought to be caused by the replacement of two amino acids that are essential for PLC activity, His356 and Tyr552, with Thr and Phe in the X and Y domain. PLC-L(2) has a wide distribution with strong expression in skeletal muscle and mapped to chromosome 3p24-25. The PH domain of PLC-L(2) bound strongly to PI(4,5)P(2) and Ins(1,4,5)P(3), and moderately to PI(4)P and PI(3,4,5)P(3). PLC-L(2) predominantly localized to perinuclear areas in both myoblast and myotube C2C12 cells. Ectopically expressed GFP-PLC-L(2) also mainly localized in perinuclear areas, including endoplasmic reticulum in COS 7 cells. Furthermore, the expression of GFP-PH showed the same intracellular distribution as the full-length PLC-L(2). All these results suggest that PLC-L(2) plays an important role in the regulation of Ins(1,4, 5)P(3) around the endoplasmic reticulum on which the Ins(1,4,5)P(3) receptor exists.

• Cocco L et al.
• Inositides in the nucleus: presence and characterisation of the isozymes of phospholipase beta family in NIH 3T3 cells.
• Biochim Biophys Acta. 1999; 1438: 295-9
• Display abstract

• Previous reports from our laboratories and others have hinted that the nucleus is a site for an autonomous signalling system acting through the activation of the inositol lipid cycle. Among phospholipases (PLC) it has been shown previously that PLCbeta1 is specifically localised in the nucleus as well as at the plasma membrane. Using NIH 3T3 cells, it has been possible to obtain, with two purification strategies, in the presence or in the absence of Nonidet P-40, both intact nuclei still maintaining the outer membrane and nuclei completely stripped of their envelope. In these nuclei, we show that not only PLCbeta1 is present, but also PLCbeta2, PLCbeta3 and PLCbeta4. The more abounding isoform is PLCbeta1 followed by PLCbeta3, PLCbeta2 and PLCbeta4, respectively. All the isoforms are enriched in nuclear preparations free from nuclear envelope and cytoplasmatic debris, indicating that the actual localisation of the PLCbeta isozymes is in the inner nuclear compartment.

• Xie Z, Bikle DD
• Phospholipase C-gamma1 is required for calcium-induced keratinocyte differentiation.
• J Biol Chem. 1999; 274: 20421-4
• Display abstract

• Phospholipase C-gamma1 is the most abundant member of the phospholipase C family in keratinocytes and is induced by calcium. Phospholipase C-gamma1, therefore, may be involved in the signal transduction system leading to calcium regulation of keratinocyte differentiation. To test this hypothesis, expression of phospholipase C-gamma1 in human keratinocytes was blocked by transfecting cells with the antisense human phospholipase C-gamma1 cDNA construct. These cells demonstrated a dramatic reduction in phospholipase C-gamma1 protein level compared with the empty vector-transfected cells and a marked reduction in the mRNA and protein levels of the differentiation markers involucrin and transglutaminase following administration of calcium. Similarly, cotransfection of antisense phospholipase C-gamma1 constructs with a luciferase reporter vector containing involucrin or transglutaminase promoters led to a substantial reduction in calcium-stimulated involucrin and transglutaminase promoter activities. Similar results were seen following treatment with a specific phospholipase C inhibitor U73122. To determine whether phospholipase C-gamma1 regulated differentiation by controlling intracellular calcium, we examined the ability of antisense phospholipase C-gamma1 to block the calcium-induced rise in intracellular calcium and found that it could. These findings indicate that phospholipase C-gamma1 is a critical component of the signaling pathway mediating calcium regulation of keratinocyte differentiation via its mobilization of intracellular calcium.

• Pawelczyk T
• Isozymes delta of phosphoinositide-specific phospholipase C.
• Acta Biochim Pol. 1999; 46: 91-8
• Display abstract

• Phospholipase C (PLC, EC 3.1.4.11) is the major starting point in the phosphatidylinositol pathway, which generates intracellular signals that regulate protein kinase C and intracellular calcium concentration. To date, three major types of phosphoinositide-specific PLC species named beta, gamma and delta, have been characterized. This article reviews recent studies on isozymes delta of PLC. Four such isozymes have been cloned and termed delta1-4. Their structural organization, regulation of activity and the interaction with membrane lipid are considered. The intracellular localization of delta isozymes and distribution in various tissues are presented. Attention is given to the pathological conditions in which an abnormal protein level of PLC delta or its activity have been observed.

• Dwivedi Y, Pandey GN
• Repeated administration of dexamethasone increases phosphoinositide-specific phospholipase C activity and mRNA and protein expression of the phospholipase C beta 1 isozyme in rat brain.
• J Neurochem. 1999; 73: 780-90
• Display abstract

• Altered hypothalamic-pituitary-adrenal (HPA) function has been shown to be associated with changes in mood and behavior. The enzyme phosphoinositide-specific phospholipase C (PI-PLC), an important component of the PI signal transduction system, plays a major role in mediating various physiological functions. In the present study, we investigated the effects of a single dose and of repeated administration (0.5 or 1.0 mg/kg for 10 days) of dexamethasone (DEX), a synthetic glucocorticoid, on PI-PLC activity and on expression of PLC isozymes (beta1, delta1, and gamma1) in rat brain. Repeated administration of DEX (1.0 mg/kg) caused a significant increase in PI-PLC activity and in protein expression of the PLC beta1 isozyme in both membrane and cytosol fractions of cortex and hippocampus; however, the repeated administration of a smaller dose of DEX (0.5 mg/kg) caused these changes only in hippocampus but not in cortex. The increase in PLC beta1 protein was associated with an increase in its mRNA level, as measured by competitive RT-PCR. A single administration of DEX (0.5 or 1.0 mg/kg) to rats had no significant effects on PI-PLC activity or on the protein expression of PLC isozymes. These results suggest that DEX up-regulates PI-PLC in rat brain, which presumably is due to a selective increase in expression of the PLC beta1 isozyme, and that these changes in PI-PLC may be related to HPA axis-mediated changes in mood and behavior.

• Carpenter G, Ji Qs
• Phospholipase C-gamma as a signal-transducing element.
• Exp Cell Res. 1999; 253: 15-24
• Display abstract

• A ubiquitous signaling event in hormonal responses is the phospholipase C (PLC)-catalyzed hydrolysis of phosphatidylinositol 4, 5-bisphosphate to produce the metabolite second messenger molecules inositol 1,4,5-trisphosphate and diacylglycerol. The former provokes a transient increase in intracellular free Ca(2+), while the latter serves as a direct activator of protein kinase C. In tyrosine kinase-dependent signaling pathways this reaction is mediated by the PLC-gamma isozymes. These are direct substrates of many tyrosine kinases in a wide variety of cell types. The mechanism of PLC-gamma activation involves its association with and phosphorylation by receptor and non-receptor tyrosine kinases, as well as interaction with specialized adaptor molecules and, perhaps, other second messenger molecules. However, the biochemistry of PLC-gamma is at a more advanced state than a clear understanding of exactly how this signaling element functions in the generation of a mitogenic response.

• Yamaga M, Fujii M, Kamata H, Hirata H, Yagisawa H
• Phospholipase C-delta1 contains a functional nuclear export signal sequence.
• J Biol Chem. 1999; 274: 28537-41
• Display abstract

• We have previously observed, using a green fluorescent protein (GFP) fusion system, that PLC-delta1 is localized mainly at the plasma membrane and in the cytosol, whereas little is present in the nucleus in Madin-Darby canine kidney cells (Fujii, M., Ohtsubo, M., Ogawa, T., Kamata, H., Hirata, H., and Yagisawa, H. (1999) Biochem. Biophys. Res. Commun. 254, 284-291). Herein, we demonstrate that PLC-delta1 has a functional nuclear export signal (NES) sequence in amino acid residues 164-177 of the EF-hand domain. The fluorescence of NES-disrupted GFP/PLC-delta1 expressed in Madin-Darby canine kidney cells was present not only at the plasma membrane and in the cytosol but also in the nucleus. Moreover, treatment with leptomycin B, a specific inhibitor of NES-dependent nuclear export, resulted in the accumulation of GFP/PLC-delta1 in the nucleus. A site-directed mutant containing a pleckstrin homology domain, which does not bind inositol 1,4,5-trisphosphate and cannot hydrolyze phosphatidylinositol 4,5-bisphosphate in vitro, accumulated in the nucleus to a much greater extent than wild-type GFP/PLC-delta1 after treatment with leptomycin B. These results suggest that PLC-delta1 is shuttled between the cytoplasm and the nucleus; its nuclear export is dependent on the leucine-rich NES sequence and its active nuclear import is regulated by an unidentified signal(s).

• Manzoli L et al.
• Nuclear phospholipase C: a novel aspect of phosphoinositide signalling.
• Anticancer Res. 1999; 19: 3753-6
• Display abstract

• The role of polyphosphoinositides in cellular signalling is well known and recently it has also been shown that the nucleus is a site for both synthesis and hydrolysis of the phosphorylated forms of phosphatidylinositol. It has been demonstrated that phospholipase C specific for inositol lipids (PLC) is one of the main steps of the inositol lipid cycle. The PLC beta family, and especially type beta 1, has given rise to considerable interest since, due to their common COOH-terminus they show nuclear localisation in addition to that at the plasma membrane. It is well established that an autonomous intranuclear inositide cycle exists, and that this cycle is endowed with conventional lipid kinases, phosphatases and PLCs. Among this latter the beta 1 type undergoes stimulation or inhibition under different stimuli and this implicates the beta 1 isoform as a key enzyme for mitogen-activated cell growth as well as for differentiation. Indeed, both the overexpression and the down-regulation of PLC beta 1, by means of antisense mRNA, have demonstrated that PLC plays a role in the nuclear compartment.

• Pawelczyk T
• [Signal transduction in the cell by phosphoinositide-specific phospholipase C isozymes]
• Postepy Hig Med Dosw. 1999; 53: 173-82
• Display abstract

• This article reviews recent studies on phosphoinositide-specific phospholipase C species named beta, gamma and delta. The localization of PLC isozymes in the cell, structural organization, regulation of PLC activity and the interaction with various receptors is presented.

• Hirose K, Kadowaki S, Tanabe M, Takeshima H, Iino M
• Spatiotemporal dynamics of inositol 1,4,5-trisphosphate that underlies complex Ca2+ mobilization patterns.
• Science. 1999; 284: 1527-30
• Display abstract

• Inositol 1,4,5-trisphosphate (IP3) is a second messenger that elicits complex spatiotemporal patterns of calcium ion (Ca2+) mobilization and has essential roles in the regulation of many cellular functions. In Madin-Darby canine kidney epithelial cells, green fluorescent protein-tagged pleckstrin homology domain translocated from the plasma membrane to the cytoplasm in response to increased concentration of IP3. The detection of translocation enabled monitoring of IP3 concentration changes within single cells and revealed spatiotemporal dynamics in the concentration of IP3 synchronous with Ca2+ oscillations and intracellular and intercellular IP3 waves that accompanied Ca2+ waves. Such changes in IP3 concentration may be fundamental to Ca2+ signaling.

• Williams RL
• Mammalian phosphoinositide-specific phospholipase C.
• Biochim Biophys Acta. 1999; 1441: 255-67
• Display abstract

• Mammalian phosphoinositide-specific phospholipases C (PI-PLCs) are involved in most receptor-mediated signal transduction pathways. The mammalian isozymes employ a modular arrangement of domains to achieve a regulated production of two key second messengers. The roles of the PH, EF hand, C2, SH2 and SH3 modules in regulation of these enzymes and in interactions with membranes and other proteins is becoming apparent from recent structural and functional studies.

• Griffith OH, Ryan M
• Bacterial phosphatidylinositol-specific phospholipase C: structure, function, and interaction with lipids.
• Biochim Biophys Acta. 1999; 1441: 237-54
• Display abstract

• The bacterial phosphatidylinositol-specific phospholipase C (PI-PLC) is a small, water-soluble enzyme that cleaves the natural membrane lipids PI, lyso-PI, and glycosyl-PI. The crystal structure, NMR and enzymatic mechanism of bacterial PI-PLCs are reviewed. These enzymes consist of a single domain folded as a (betaalpha)(8)-barrel (TIM barrel), are calcium-independent, and interact weakly with membranes. Sequence similarity among PI-PLCs from different bacterial species is extensive, and includes the residues involved in catalysis. Bacterial PI-PLCs are structurally similar to the catalytic domain of mammalian PI-PLCs. Comparative studies of both prokaryotic and eukaryotic isozymes have proved useful for the identification of distinct regions of the proteins that are structurally and functionally important.

• Medvedeva ND
• [Phosphoinositide-specific phospholipase C gamma 1--a key protein for signal transduction, triggered by growth factors]
• Tsitologiia. 1999; 41: 997-1005
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• The review is devoted to the participation of phospholipase C gamma 1 in cell signaling. A great attention is paid to mechanisms of activation of phospholipase C gamma and its interaction with other signaling molecules. The role of phospholipase C gamma binding to cytoskeletal elements in cell signaling is discussed.

• Yagisawa H, Fujii M, Hirata M
• Phospholipase C-delta and related molecules.
• Biochem Soc Trans. 1999; 27: 652-7

• Hitomi T, Yanagi S, Inatome R, Yamamura H
• Cross-linking of the B cell receptor induces activation of phospholipase D through Syk, Btk and phospholipase C-gamma2.
• FEBS Lett. 1999; 445: 371-4
• Display abstract

• Phospholipase D (PLD) has been proposed to play a key role in the signal transduction of cellular responses to various extracellular signals. Herein we provide biochemical and genetic evidence that cross-linking of the B cell receptor (BCR) induces rapid activation of PLD through a Syk-, Btk- and phospholipase C (PLC)-gamma2-dependent pathway in DT40 cells. Activation of PLD upon BCR engagement is completely blocked in Syk- or Btk-deficient cells, but unaffected in Lyn-deficient cells. Furthermore, in PLC-gamma2-deficient cells, BCR engagement failed to activate PLD. These results demonstrate that Syk, Btk and PLC-gamma2 are essential for BCR-induced PLD activation.

• Sandnes D, Dajani O, Bjorneby A, Christoffersen T
• The relationship between activation of phosphoinositide-specific phospholipase C and growth stimulation by Ca2+-mobilizing hormones in hepatocytes.
• Pharmacol Toxicol. 1999; 84: 234-40
• Display abstract

• Previous studies have shown that while vasopressin and angiotensin II are markedly more effective than norepinephrine and prostaglandin F2alpha in eliciting an acute elevation of inositol 1,4,5-trisphosphate (IP3), norepinephrine and prostaglandin F2alpha produce larger enhancement of DNA synthesis. This suggests that the initial activation of phosphoinositide-specific phospholipase C is not a common factor for the growth response to these agonists, but does not exclude a role of the integral of phospholipase C activity over a prolonged part of the prereplicative period, during which agonist-specific changes in responsiveness might occur. We show that vasopressin and angiotensin II also cause a prolonged elevation of cellular IP3 levels. which remain elevated for at least 60 min., while norepinephrine and prostaglandin F2alpha elevate IP3 levels slightly and transiently For vasopressin the dose-effect curves for IP3 accumulation and stimulation of DNA synthesis were closely parallel, while this was not the case for angiotensin II, norepinephrine, or prostaglandin F2alpha. After cultivation of the hepatocytes, hormone-stimulated IP3 accumulation rapidly declined, particularly in response to norepinephrine and prostaglandin F2alpha. When the IP3 response to norepinephrine and prostaglandin F2alpha was completely down-regulated, these agonists still enhanced the DNA synthesis. These results suggest that other mechanisms in addition to IP3 accumulation and Ca2+ release are likely to be involved in the growth stimulatory effects of the Ca2+-mobilizing agonists studied here, in particular for angiotensin II, norepinephrine, and prostaglandin F2alpha.

• Perera IY, Heilmann I, Boss WF
• Transient and sustained increases in inositol 1,4,5-trisphosphate precede the differential growth response in gravistimulated maize pulvini.
• Proc Natl Acad Sci U S A. 1999; 96: 5838-43
• Display abstract

• The internodal maize pulvinus responds to gravistimulation with differential cell elongation on the lower side. As the site of both graviperception and response, the pulvinus is an ideal system to study how organisms sense changes in orientation. We observed a transient 5-fold increase in inositol 1,4,5-trisphosphate (IP3) within 10 s of gravistimulation in the lower half of the pulvinus, indicating that the positional change was sensed immediately. Over the first 30 min, rapid IP3 fluctuations were observed between the upper and lower halves. Maize plants require a presentation time of between 2 and 4 h before the cells on the lower side of the pulvinus are committed to elongation. After 2 h of gravistimulation, the lower half consistently had higher IP3, and IP3 levels on the lower side continued to increase up to approximately 5-fold over basal levels before visible growth. As bending became visible after 8-10 h, IP3 levels returned to basal values. Additionally, phosphatidylinositol 4-phosphate 5-kinase activity in the lower pulvinus half increased transiently within 10 min of gravistimulation, suggesting that the increased IP3 production was accompanied by an up-regulation of phosphatidylinositol 4, 5-bisphosphate biosynthesis. Neither IP3 levels nor phosphatidylinositol 4-phosphate 5-kinase activity changed in pulvini halves from vertical control plants. Our data indicate the involvement of IP3 and inositol phospholipids in both short- and long-term responses to gravistimulation. As a diffusible second messenger, IP3 provides a mechanism to transmit and amplify the signal from the perceiving to the responding cells in the pulvinus, coordinating a synchronized growth response.

• Heinz DW, Essen LO, Williams RL
• Structural and mechanistic comparison of prokaryotic and eukaryotic phosphoinositide-specific phospholipases C.
• J Mol Biol. 1998; 275: 635-50
• Display abstract

• Phosphoinositide-specific phospholipases C (PI-PLCs) are ubiquitous enzymes that catalyse the hydrolysis of phosphoinositides to inositol phosphates and diacylglycerol (DAG). Whereas the eukaryotic PI-PLCs play a central role in most signal transduction cascades by producing two second messengers, inositol-1,4,5-trisphosphate and DAG, prokaryotic PI-PLCs are of interest because they act as virulence factors in some pathogenic bacteria. Bacterial PI-PLCs consist of a single domain of 30 to 35 kDa, while the much larger eukaryotic enzymes (85 to 150 kDa) are organized in several distinct domains. The catalytic domain of eukaryotic PI-PLCs is assembled from two highly conserved polypeptide stretches, called regions X and Y, that are separated by a divergent linker sequence. There is only marginal sequence similarity between the catalytic domain of eukaryotic and prokaryotic PI-PLCs. Recently the crystal structures of a bacterial and a eukaryotic PI-PLC have been determined, both in complexes with substrate analogues thus enabling a comparison of these enzymes in structural and mechanistic terms. Eukaryotic and prokaryotic PI-PLCs contain a distorted (beta alpha)8-barrel as a structural motif with a surprisingly large structural similarity for the first half of the (beta alpha)8-barrel and a much weaker similarity for the second half. The higher degree of structure conservation in the first half of the barrel correlates with the presence of all catalytic residues, in particular two catalytic histidine residues, in this portion of the enzyme. The second half contributes mainly to the features of the substrate binding pocket that result in the distinct substrate preferences exhibited by the prokaryotic and eukaryotic enzymes. A striking difference between the enzymes is the utilization of a catalytic calcium ion that electrostatically stabilizes the transition state in eukaryotic enzymes, whereas this role is filled by an analogously positioned arginine in bacterial PI-PLCs. The catalytic domains of all PI-PLCs may share not only a common fold but also a similar catalytic mechanism utilizing general base/acid catalysis. The conservation of the topology and parts of the active site suggests a divergent evolution from a common ancestral protein.

• Hodson EA, Ashley CC, Hughes AD, Lymn JS
• Regulation of phospholipase C delta 1 activity by GTP-binding proteins: rhoA as an inhibitory modulator.
• Biochem Soc Trans. 1998; 26: 128-128

• Naylor CE et al.
• Structure of the key toxin in gas gangrene.
• Nat Struct Biol. 1998; 5: 738-46
• Display abstract

• Clostridium perfringens alpha-toxin is the key virulence determinant in gas gangrene and has also been implicated in the pathogenesis of sudden death syndrome in young animals. The toxin is a 370-residue, zinc metalloenzyme that has phospholipase C activity, and can bind to membranes in the presence of calcium. The crystal structure of the enzyme reveals a two-domain protein. The N-terminal domain shows an anticipated structural similarity to Bacillus cereus phosphatidylcholine-specific phospholipase C (PC-PLC). The C-terminal domain shows a strong structural analogy to eukaryotic calcium-binding C2 domains. We believe this is the first example of such a domain in prokaryotes. This type of domain has been found to act as a phospholipid and/or calcium-binding domain in intracellular second messenger proteins and, interestingly, these pathways are perturbed in cells treated with alpha-toxin. Finally, a possible mechanism for alpha-toxin attack on membrane-packed phospholipid is described, which rationalizes its toxicity when compared to other, non-haemolytic, but homologous phospholipases C.

• Kanno T
• [Exocrine pancreas]
• Tanpakushitsu Kakusan Koso. 1998; 43: 1794-9

• Mosior M, Six DA, Dennis EA
• Group IV cytosolic phospholipase A2 binds with high affinity and specificity to phosphatidylinositol 4,5-bisphosphate resulting in dramatic increases in activity.
• J Biol Chem. 1998; 273: 2184-91
• Display abstract

• The group IV cytosolic phospholipase A2 (cPLA2) exhibits a potent and specific increase in affinity for lipid surfaces containing phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) at physiologically relevant concentrations. Specifically, the presence of 1 mol% PtdIns(4,5)P2 in phosphatidylcholine vesicles results in a 20-fold increase in the binding affinity of cPLA2. This increased affinity is accompanied by an increase in substrate hydrolysis of a similar magnitude. The binding studies and kinetic analysis indicate that PtdIns(4,5)P2 binds to cPLA2 in a 1:1 stoichiometry. The magnitude of the effect of PtdIns(4,5)P2 is unique among anionic phospholipids and larger than that for other polyphosphate phosphatidylinositols. The effect of PtdIns(4,5)P2 on the activity of cPLA2 is at least an order of magnitude larger than the concomitant changes in the fraction of the enzyme associated with lipid membranes. Striking parallels between the interaction of cPLA2 with PtdIns(4,5)P2 and the interaction of the pleckstrin homology domain of phospholipase C delta 1 with PtdIns(4,5)2 combined with sequence analysis of cPLA2 lead us to propose the existence and location of a pleckstrin homology domain in cPLA2. We further show that the very nature of the interaction of proteins such as cPLA2 with multiple ligands incorporated into membranes follows a specific model which necessitates the use of an experimental methodology suitable for a membrane interface to allow for a meaningful analysis of the data.

• Iwasaki Y et al.
• Two distinct phosphatidylinositol-specific phospholipase Cs from Streptomyces antibioticus.
• Biochim Biophys Acta. 1998; 1391: 52-66
• Display abstract

• Two phosphatidylinositol-specific phospholipase C (PI-PLC) genes from Streptomyces antibioticus were cloned by a shotgun method using Streptomyces lividans TK24 as a host. The genes of the two PI-PLCs (named as PLC1 and PLC2) were adjoined and opposite in the direction of transcription/translation. Both of them were confirmed to be expressed in S. antibioticus. The two enzymes were different in the following properties. (i) PLC2 had considerable sequence similarity to other bacterial PI-PLCs, while PLC1 had a short stretch that was similar to PI-PLCs of eukaryotes rather than the other bacterial enzymes. (ii) PLC1 was Ca2+-dependent, whereas PLC2 was not. (iii) PLC1 generated myo-inositol-1-phosphate and myo-inositol-1:2-cyclic phosphate simultaneously from PI, but PLC2 showed sequential formation of them. (iv) PLC2 has GPI-anchor-degrading activity while PLC1 does not have. Both enzymes did not hydrolyze phosphatidylcholine, phosphatidylinositol-4-monophosphate and phosphatidylinositol-4,5-bisphosphate. Both PLC1 and PLC2 contained two histidine residues that might be catalytic residues. PLC1 has residues that possibly form a Ca2+-binding site. Then it was suggested that both PLC1 and PLC2 act according to the catalytic mechanism using the two histidine residues as proposed in both eukaryotic and prokaryotic enzymes, but that PLC1 has a more 'eukaryotic' mechanism in which Ca2+ participates than that of the Ca2+-independent bacterial enzymes. Thus, we propose that PLC2 is a conventional 'bacteria-type' enzyme, while PLC1 is more closely related to the eukaryotic enzymes rather than the bacterial enzymes.

• Bae YS, Cantley LG, Chen CS, Kim SR, Kwon KS, Rhee SG
• Activation of phospholipase C-gamma by phosphatidylinositol 3,4,5-trisphosphate.
• J Biol Chem. 1998; 273: 4465-9
• Display abstract

• Signal transduction across cell membranes often involves the activation of both phosphatidylinositol (PI)-specific phospholipase C (PLC) and phosphoinositide 3-kinase (PI 3-kinase). Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), a substrate for both enzymes, is converted to phosphatidylinositol 3,4, 5-trisphosphate (PI(3,4,5)P3) by the action of PI 3-kinase. Here, we show that PI(3,4,5)P3 activates purified PLC-gamma isozymes by interacting with their Src homology 2 domains. Furthermore, the expression of an activated catalytic subunit of PI 3-kinase in COS-7 cells resulted in an increase in inositol phosphate formation, whereas platelet-derived growth factor-induced PLC activation in NIH 3T3 cells was markedly inhibited by the specific PI 3-kinase inhibitor LY294002. These results suggest that receptors coupled to PI 3-kinase may activate PLC-gamma isozymes indirectly, in the absence of PLC-gamma tyrosine phosphorylation, through the generation of PI(3,4,5)P3.

• Hendrickson HS, Hendrickson EK
• A thiophosphate analog of dimyristoylphosphatidyl-inositol-4-phosphate is a substrate for mammalian phosphoinositide-specific phospholipase C.
• Bioorg Med Chem Lett. 1998; 8: 1057-60
• Display abstract

• 1,2-Dimyristoyloxypropane-3-thiophosphate(rac-1-myo-inositol-4- phosphate), a thiophosphate analog of dimyristoyl phosphatidylinositol-4-phosphate was synthesized as a substrate for mammalian phosphoinositide-specific phospholipase C. Its activity with delta(1-132)-PI-PLC-delta 1 (a deletion mutant with the N-terminal pleckstrin homology domain removed) was studied in sonicated dispersions, with and without added Triton X-100. It had an initial activity of about 30 mumol min-1 mg-1, which rapidly decreased due to substrate depletion in the vesicle or micelle. The slower rate of hydrolysis appeared limited by enzyme hopping or exchange of substrate between vesicles or micelles, which was more rapid in the presence of detergent.

• Tan CA, Roberts MF
• Engineering of the nonspecific phospholipase C from Bacillus cereus: replacement of glutamic acid-4 by alanine results in loss of interfacial catalysis and enhanced phosphomonoesterase activity.
• Biochemistry. 1998; 37: 4275-9
• Display abstract

• The nonspecific phospholipase C from Bacillus cereus is a zinc metalloenzyme that catalyzes the hydrolysis of phospholipids to yield diacylglycerol and a phosphate monoester. Glu-4 has been proposed as a potential candidate for the general base in the hydrolysis reaction and was shown to interact with the substrate headgroup. Site-specific mutagenesis studies suggest that Glu-4 is important for substrate binding but not for catalysis. This residue is also critical for the enzyme's preference for a phosphodiester substrate. PA, both monomeric and micellar, is shown to be a poor substrate and inhibitor of wild-type PLC. When Glu-4 was mutated to an alanine, a significant increase in PA hydrolysis and a decrease in PC hydrolysis were observed. Unlike the wild type, kinetic studies suggest that the Glu-4-->Ala mutant does not exhibit interfacial activation and processive catalysis. Glu-4 is part of a highly flexible loop flanking the entrance to the active site, suggesting that this loop might constitute an interfacial binding recognition site. This is the first evidence for the presence of an interfacial binding site distinct from the active site in the nonspecific PLC.

• Di Pietro R, Rana R
• Age-related defect of phospholipase C activity, differential expression of the beta 2 isoform in active T lymphocytes from aged humans.
• Hum Immunol. 1998; 59: 25-8
• Display abstract

• The activation of phosphoinositide-specific PLC is one of the early cellular responses to various growth factors and mitogens. It is known that these functions are altered with ageing. Here we show that the beta 2 isozyme is decreased with ageing in a peculiar T lymphocyte subpopulation involved in the immune response, namely the active T lymphocytes. The presence and cellular distribution of PLC isozymes were investigated with immunochemical and immunocytochemical methods. The in situ immunocytochemistry displayed the presence of the beta and gamma isoforms in the cytoplasm, while no reactivity for the delta isoform was observed regardless of the age. The immunoblot analysis showed an increased expression in the beta 2 isoform in the young and an equal expression of the gamma 1 isoform in either age group. Our findings suggest that the age-related defect of PLC activity is possibly due to an impaired expression of isozymes in aged active T lymphocytes.

• Mitchell J, Mayeenuddin LH
• Purification, G protein activation, and partial amino acid sequence of a novel phospholipase C from squid photoreceptors.
• Biochemistry. 1998; 37: 9064-72
• Display abstract

• Invertebrate visual signal transduction is initiated by rhodopsin activation of a guanine nucleotide binding protein, Gq, which stimulates phospholipase C (PLC) activity. We have previously purified a 140-kDa PLC enzyme from squid photoreceptors that is regulated by squid Gq. In these studies, an additional PLC enzyme was purified from the cytosol of squid photoreceptors and identified as a 70-kDa protein by SDS-polyacrylamide gel electrophoresis. Hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) by PLC-70 was optimal at pH 5 in the presence of 100 microM Ca2+ with a specific activity of 10.3 micromol min-1 mg-1. A polyclonal antibody raised against purified PLC-70 did not recognize purified PLC-140, and proteolytic digestion of the two purified enzymes with trypsin or Staphylococcus aureaus V8 protease showed distinct patterns of peptide fragments, indicating that PLC-70 is not a fragment of PLC-140. The partial amino acid sequence of the protein showed homology with PLC21 and norpA isozymes cloned from Drosophila, and mammalian PLC beta isozymes. Reconstitution of purified GTPgammaS-bound soluble squid Gq with PLC-70 resulted in significant enhancement of PIP2 hydrolysis over a range of Ca2+ concentrations and shifted the maximum activation by calcium to 1 microM. These results suggest that cephalopod phototransduction is mediated by Gq activation of more than one cytosolic PLC enzyme.

• Shimohama S et al.
• Phospholipase C isozymes in the human brain and their changes in Alzheimer's disease.
• Neuroscience. 1998; 82: 999-1007
• Display abstract

• Phosphoinositide-specific phospholipase C is a key enzyme in signal transduction. We have previously demonstrated that an isozyme of phospholipase C, phospholipase C-delta1, accumulates aberrantly in the brains of patients with Alzheimer's disease. In the present study, we examined the property of phospholipase C isozymes in human brains using the methods of chromatofocusing and gel filtration chromatography, and investigated their changes in Alzheimer's disease brains. The chromatofocusing profile of human brain phospholipase C activity on a Mono P HR column demonstrated that phospholipase C-gamma1, exhibiting an isoelectric point value of 5.2, and phospholipase C-delta1, exhibiting isoelectric point values of 5.2 and 4.6, are partly overlapped in their elution. In contrast, the elution profiles of control and Alzheimer's disease brain phospholipase C on Superdex 200 pg column gel filtration chromatography indicated that phospholipase C-gamma1 and phospholipase C-delta1 can be separated with the elution position having a molecular weight of about 240,000 and 140,000, respectively, in the human brain. Using this gel filtration chromatography it was revealed that the phospholipase C-gamma1 activity was significantly decreased and the phospholipase C-delta1 activity was significantly increased in Alzheimer's disease brains compared with controls. These results suggest that the phospholipase C isozymes are differentially involved in Alzheimer's disease.

• Miyamoto A, Ohshika H
• Molecular diversity and double regulatory mechanism of activation of phospholipase C in rat brain.
• Life Sci. 1998; 62: 1549-53
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• Whereas evidence for a G protein-dependent stimulation of phospholipase C (PLC) is abundant, reports on the inhibition of PLC through a G protein-mediated pathway have only recently begun to appear. In the present study, cerebral cortex membranes were chosen since they have a readily measurable Gpp[NH]p and Ca2+-stimulated PLC activity. Nanomolar concentrations of Gpp[NH]p, a hydrolysis-resistant GTP analogue, inhibited basal inositol 1,4,5-trisphosphate (IP3) production, with a maximum inhibition of 25% at 10 nM. Increasing the concentrations of Gpp[NH]p to over 10 nM resulted in a reversal of the inhibitory effect and onset of stimulation of IP3 production. GDPbetaS as a G protein inhibitor and U-73122 as a putative PLC-beta inhibitor had little effect on basal IP3 production at 100 microM and 1 microM, respectively. However, GDPbetaS and U-73122 completely antagonized both the inhibition and the stimulation of IP3 production produced by lower and higher concentrations, respectively, of Gpp[NH]p. Rat cortical membranes expressed a greater amount of PLC-beta1. These data suggest that PLC-beta1 isozymes may be regulated by both inhibitory and stimulatory G protein-mediated mechanisms.

• Bahk YY et al.
• Localization of two forms of phospholipase C-beta1, a and b, in C6Bu-1 cells.
• Biochim Biophys Acta. 1998; 1389: 76-80
• Display abstract

• Phospholipase C-beta1 (PLC-beta1), one of the PLC-beta isozymes, exists as two immunologically distinguishable polypeptides of 150 (PLC-beta1a) and 140 kDa (PLC-beta1b) which are encoded in two distinct transcripts and generated by alternative splicing of a single gene. In this study, the subcellular localization of the two phospholipases C-beta1 proteins was examined in rat C6Bu-1 glioma cells using immunological techniques. Immunoblot analysis revealed that the two forms of PLC-beta1 were detectable in both cytosolic and nuclear fractions. PLC-beta1a appeared to be located preferentially in the cytosol, whereas PLC-beta1b was found predominantly in the nuclei of C6Bu-1 cells. Immunocytochemical experiments confirmed the differential localization of the two PLC-beta1 species in C6Bu-1 cells. These results suggest that the two PLC-beta1 proteins may have different physiological roles in the cell.

• Katan M
• Families of phosphoinositide-specific phospholipase C: structure and function.
• Biochim Biophys Acta. 1998; 1436: 5-17
• Display abstract

• A large number of extracellular signals stimulate hydrolysis of phosphatidylinositol 4,5-bisphosphate by phosphoinositide-specific phospholipase C (PI-PLC). PI-PLC isozymes have been found in a broad spectrum of organisms and although they have common catalytic properties, their regulation involves different signalling pathways. A number of recent studies provided an insight into domain organisation of PI-PLC isozymes and contributed towards better understanding of the structural basis for catalysis, cellular localisation and molecular changes that could underlie the process of their activation.

• Hondal RJ et al.
• Mechanism of phosphatidylinositol-specific phospholipase C: a unified view of the mechanism of catalysis.
• Biochemistry. 1998; 37: 4568-80
• Display abstract

• The mechanism of phosphatidylinositol-specific phospholipase C (PI-PLC) has been suggested to resemble that of ribonuclease A. The goal of this work is to rigorously evaluate the mechanism of PI-PLC from Bacillus thuringiensis by examining the functional and structural roles of His-32 and His-82, along with the two nearby residues Asp-274 and Asp-33 (which form a hydrogen bond with His-32 and His-82, respectively), using site-directed mutagenesis. In all, twelve mutants were constructed, which, except D274E, showed little structural perturbation on the basis of 1D NMR and 2D NOESY analyses. The H32A, H32N, H32Q, H82A, H82N, H82Q, H82D, and D274A mutants showed a 10(4)-10(5)-fold decrease in specific activity toward phosphatidylinositol; the D274N, D33A, and D33N mutants retained 0. 1-1% activity, whereas the D274E mutant retained 13% activity. Steady-state kinetic analysis of mutants using (2R)-1, 2-dipalmitoyloxypropane-3-(thiophospho-1d-myo-inositol) (DPsPI) as a substrate generally agreed well with the specific activity toward phosphatidylinositol. The results suggest a mechanism in which His-32 functions as a general base to abstract the proton from 2-OH and facilitates the attack of the deprotonated 2-oxygen on the phosphorus atom. This general base function is augmented by the carboxylate group of Asp-274 which forms a diad with His-32. The H82A and D33A mutants showed an unusually high activity with substrates featuring low pKa leaving groups, such as DPsPI and p-nitrophenyl inositol phosphate (NPIPs). These results suggest that His-82 functions as the general acid with assistance from Asp-33, facilitating the departure of the leaving group by protonation of the glycerol O3 oxygen. The Bronsted coefficients obtained for the WT and the D33N mutant indicate a high degree of proton transfer to the leaving group and further underscore the "helper" function of Asp-33. The complete mechanism also includes activation of the phosphate group toward nucleophilic attack by a hydrogen bond between Arg-69 and a nonbridging oxygen atom. The overall mechanism can be described as "complex" general acid-general base since three elements are required for efficient catalysis.

• Wakelam MJ, Pettitt TR
• Determination of phospholipase C- or phospholipase D-catalyzed phosphatidylcholine hydrolysis.
• Methods Mol Biol. 1998; 105: 141-9

• Perisic O, Fong S, Lynch DE, Bycroft M, Williams RL
• Crystal structure of a calcium-phospholipid binding domain from cytosolic phospholipase A2.
• J Biol Chem. 1998; 273: 1596-604
• Display abstract

• Cytosolic phospholipase A2 (cPLA2) is a calcium-sensitive 85-kDa enzyme that hydrolyzes arachidonic acid-containing membrane phospholipids to initiate the biosynthesis of eicosanoids and platelet-activating factor, potent inflammatory mediators. The calcium-dependent activation of the enzyme is mediated by an N-terminal C2 domain, which is responsible for calcium-dependent translocation of the enzyme to membranes and that enables the intact enzyme to hydrolyze membrane-resident substrates. The 2.4-A x-ray crystal structure of this C2 domain was solved by multiple isomorphous replacement and reveals a beta-sandwich with the same topology as the C2 domain from phosphoinositide-specific phospholipase C delta 1. Two clusters of exposed hydrophobic residues surround two adjacent calcium binding sites. This region, along with an adjoining strip of basic residues, appear to constitute the membrane binding motif. The structure provides a striking insight into the relative importance of hydrophobic and electrostatic components of membrane binding for cPLA2. Although hydrophobic interactions predominate for cPLA2, for other C2 domains such as in "conventional" protein kinase C and synaptotagmins, electrostatic forces prevail.

• Grobler JA, Hurley JH
• Catalysis by phospholipase C delta1 requires that Ca2+ bind to the catalytic domain, but not the C2 domain.
• Biochemistry. 1998; 37: 5020-8
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• The hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) by phosphoinositide-specific phospholipase C (PLC) is absolutely dependent on Ca2+. The PH domain truncated catalytic core of rat phospholipase C delta1 (PLC-delta1) has Ca2+ binding sites in its catalytic and C2 domains, and potential Ca2+ binding sites in two EF-hands. A catalytically inactive PLC-delta1 catalytic core bound with low affinity to PIP2-containing vesicles in the presence of Ca2+. A mutant PLC-delta1 has been engineered which lacks the C2 domain Ca2+ binding site and the surrounding loops known as the jaws. Isothermal calorimetric titration showed four Ca2+ ions bind to the wild-type PLC-delta1 catalytic core in solution but only one binds to the C2 domain jaws deletion mutant. The activity and Ca2+ dependence of wild-type and mutant phospholipase Cs were determined using substrate incorporated in detergent micelles and in large unilamellar vesicles. The activities of wild-type and mutant were identical to each other in both assay systems. Wild-type and the C2 jaws deletion mutant of PLC have Hill coefficients of 1.12-1.16 with respect to [Ca2+]. We conclude that a single Ca2+ bound to the catalytic domain is entirely responsible for the Ca2+ dependence of the basal activity of PLC-delta1.

• James S
• The structure of phospholipase C isoforms and the regulation of phosphoinositide hydrolysis.
• Biochem Soc Trans. 1998; 26: 354-9

• Hendrickson HS
• Continuous spectrophotometric assay of mammalian phosphoinositide-specific phospholipase Cdelta1 with a thiophosphate substrate analog.
• Biochim Biophys Acta. 1998; 1392: 16-22
• Display abstract

• 1,2-Dimyristoyloxypropane-3-thiophospho(1D-1-myo-inositol) (D-thio-DMPI) was used as a substrate for the continuous assay of phosphoinositide-specific phospholipase C (PI-PLC). Its activity with a Delta(1-132) deletion mutant of mammalian PI-PLCdelta1 is about one-fourth that with PI under similar conditions. Optimal conditions for the assay include 0.2 mM substrate, 0.2 mM Ca2+, and a mole ratio of hexadecylphosphocholine detergent to substrate of 2.0. A minimum of about 60 ng of pure enzyme can be detected. The apparent bulk Km for PI-PLC with D-thio-DMPI under these conditions is about 6 microM. Enzyme activity as a function of surface concentration of substrate shows no sign of saturation up to the maximum mole fraction.

• Kopka J, Pical C, Gray JE, Muller-Rober B
• Molecular and enzymatic characterization of three phosphoinositide-specific phospholipase C isoforms from potato.
• Plant Physiol. 1998; 116: 239-50
• Display abstract

• Many cellular responses to stimulation of cell-surface receptors by extracellular signals are transmitted across the plasma membrane by hydrolysis of phosphatidylinositol-4,5-bisphosphate (PIP2), which is cleaved into diacylglycerol and inositol-1,4,5-trisphosphate by phosphoinositide-specific phospholipase C (PI-PLC). We present structural, biochemical, and RNA expression data for three distinct PI-PLC isoforms, StPLC1, StPLC2, and StPLC3, which were cloned from a guard cell-enriched tissue preparation of potato (Solanum tuberosum) leaves. All three enzymes contain the catalytic X and Y domains, as well as C2-like domains also present in all PI-PLCs. Analysis of the reaction products obtained from PIP2 hydrolysis unequivocally identified these enzymes as genuine PI-PLC isoforms. Recombinant StPLCs showed an optimal PIP2-hydrolyzing activity at 10 microM Ca2+ and were inhibited by Al3+ in equimolar amounts. In contrast to PI-PLC activity in plant plasma membranes, however, recombinant enzymes could not be activated by Mg2+. All three stplc genes are expressed in various tissues of potato, including leaves, flowers, tubers, and roots, and are affected by drought stress in a gene-specific manner.

• Falasca M, Logan SK, Lehto VP, Baccante G, Lemmon MA, Schlessinger J
• Activation of phospholipase C gamma by PI 3-kinase-induced PH domain-mediated membrane targeting.
• EMBO J. 1998; 17: 414-22
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• Signaling via growth factor receptors frequently results in the concomitant activation of phospholipase C gamma (PLC gamma) and phosphatidylinositol (PI) 3-kinase. While it is well established that tyrosine phosphorylation of PLC gamma is necessary for its activation, we show here that PLC gamma is regulated additionally by the lipid products of PI 3-kinase. We demonstrate that the pleckstrin homology (PH) domain of PLC gamma binds to phosphatidylinositol 3,4,5-trisphosphate [PdtIns(3,4,5)P3], and is targeted to the membrane in response to growth factor stimulation, while a mutated version of this PH domain that does not bind PdtIns(3,4,5)P3 is not membrane targeted. Consistent with these observations, activation of PI 3-kinase causes PLC gamma PH domain-mediated membrane targeting and PLC gamma activation. By contrast, either the inhibition of PI 3-kinase by overexpression of a dominant-negative mutant or the prevention of PLC gamma membrane targeting by overexpression of the PLC gamma PH domain prevents growth factor-induced PLC gamma activation. These experiments reveal a novel mechanism for cross-talk and mutual regulation of activity between two enzymes that participate in the control of phosphoinositide metabolism.

• Zhou C, Roberts MF
• Nonessential activation and competitive inhibition of bacterial phosphatidylinositol-specific phospholipase C by short-chain phospholipids and analogues.
• Biochemistry. 1998; 37: 16430-9
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• Phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus thuringiensis is an allosteric enzyme with both a phospholipid activator site and an active site. The activation of PI-PLC enzyme is optimal with phosphatidylcholine (PC) binding to the activator site and anchoring the enzyme to the interface [Zhou, C., et al. (1997) Biochemistry 36, 347-355; Zhou, C., et al. (1997) Biochemistry 36, 10089-10091]. In contrast to PC, anionic short-chain phospholipids with smaller headgroups [phosphatidylmethanol (PMe) and phosphatidic acid (PA)] as well as phosphatidylglycerol (PG) can bind to both sites playing dual roles: nonessential activation and competitive inhibition of cyclic-(1, 2)-inositol phosphate hydrolysis. PG is also a substrate, albeit a poor one, for PI-PLC, and is cleaved slowly to form alpha-glycerol phosphate. Analysis of enzyme kinetics using cIP as the substrate coupled with effects of different short-chain phospholipids on enzyme intrinsic fluorescence indicates that anionic phospholipids with small headgroups bind to the two sites with different affinities. If no interface is present, all dihexanoylphospholipids bind to the activator site more strongly than to the active site. When the activator site is occupied, it is likely that the enzyme undergoes a conformational change that allows phospholipids to bind easily to the active site. Such behavior is consistent with the observation that enzyme activation is detected at low short-chain anionic phospholipid concentrations with inhibition observed at higher concentrations, and that only inhibition is seen with these phospholipids added as monomers in the presence of a PC interface that optimally activates the PI-PLC. A kinetic model is used to extract the affinity of short-chain lipids for the active site from experimental data.

• Shimohama S et al.
• Differential expression of rat brain phospholipase C isozymes in development and aging.
• Biochem Biophys Res Commun. 1998; 243: 210-6
• Display abstract

• Phosphoinositide-specific phospholipase C (PLC) is a key enzyme in signal transduction. In the present study we examined developmental and aging changes in three PLC isozymes (beta 1, gamma 1, and delta 1) in the rat brain. Enzyme assays and immunoblot analyses after gel filtration chromatography of brain extracts from embryonic day 19 and postnatal 4- and 48-week rats indicated that gamma-specific activity was highest in fetal brain and decreased with aging, that beta 1-specific activity was high at 4 weeks but essentially undetected in fetal brain, and that delta 1-specific activity was high at both 4 and 48 weeks with faint detection in fetal brain. Our results suggest that the gamma 1 isozyme may be particularly involved in cell division and growth during the histo-genesis of the central nervous system, while beta 1 and delta 1 isozymes may take part in processes of its maturation and maintenance.

• Hirayama T, Mitsukawa N, Shibata D, Shinozaki K
• AtPLC2, a gene encoding phosphoinositide-specific phospholipase C, is constitutively expressed in vegetative and floral tissues in Arabidopsis thaliana.
• Plant Mol Biol. 1997; 34: 175-80
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• A cDNA encoding a phosphoinositide-specific phospholipase C (PI-PLC) from the higher plant Arabidopsis thaliana was cloned and characterized. The gene corresponding to this cDNA is designated AtPLC2. The overall structure of the predicted AtPLC2 protein is similar to those of plant PI-PLCs and mammalian delta-type PI-PLCs. Northern blot analysis revealed that AtPLC2 is expressed constitutively whereas AtPLC1S, another gene for PI-PLC of Arabidopsis, is induced by environmental stresses such as dehydration and salinity, indicating that the function of AtPLC2 is distinct from that of AtPLC1S. The AtPLC2 mRNA was detected in vegetative and floral tissues. We determined the positions of these two PI-PLCs genes on Arabidopsis chromosomes by RFLP mapping using P1 genomic clones.

• Moser J, Gerstel B, Meyer JE, Chakraborty T, Wehland J, Heinz DW
• Crystal structure of the phosphatidylinositol-specific phospholipase C from the human pathogen Listeria monocytogenes.
• J Mol Biol. 1997; 273: 269-82
• Display abstract

• The X-ray crystal structure of the phosphatidylinositol-specific phospholipase C (PI-PLC) from the human pathogen Listeria monocytogenes has been determined both in free form at 2.0 A resolution, and in complex with the competitive inhibitor myo-inositol at 2.6 A resolution. The structure was solved by a combination of molecular replacement using the structure of Bacillus cereus PI-PLC and single isomorphous replacement. The enzyme consists of a single (beta alpha)8-barrel domain with the active site located at the C-terminal side of the beta-barrel. Unlike other (beta alpha)8-barrels, the barrel in PI-PLC is open because it lacks hydrogen bonding interactions between beta-strands V and VI. myo-Inositol binds to the active site pocket by making specific hydrogen bonding interactions with a number of charged amino acid side-chains as well as a coplanar stacking interaction with a tyrosine residue. Despite a relatively low sequence identity of approximately 24%, the structure is highly homologous to that of B.cereus PI-PLC with an r.m.s. deviation for 228 common C alpha positions of 1.46 A. Larger differences are found for loop regions that accommodate most of the numerous amino acid insertions and deletions. The active site pocket is also well conserved with only two amino acid replacements directly implicated in inositol binding.

• Nebigil CG
• Suppression of phospholipase C beta, gamma, and delta families alters cell growth and phosphatidylinositol 4,5-bisphosphate levels.
• Biochemistry. 1997; 36: 15949-58
• Display abstract

• Phosphatidylinositol-specific phospholipase C (PLC) activity reflects a summation of the activities of three families, beta, gamma, and delta, each of which is regulated differently. In order to understand the contribution of each family to cell proliferation signaling, expression of each family was suppressed by use of an inducible expression vector for antisense PLC sequences in a single cell line, FTO-2B rat hepatocytes. Activation of second messengers of PLC [diacylglycerol (DAG) and inositol 1,4,5-tris(phosphate) (IP3)] was dramatically reduced, providing a strategy for probing the consequences of PLC deficiency on cell function. Importantly, while one PLC family was suppressed, the other PLCs actively responded to specific stimuli, suggesting parallel and independent signaling pathways for each PLC family in FTO-2B cells. Selective suppression of each PLC family altered cell growth markedly and differentially. The rank order for suppression of cell growth by loss of a PLC family was gamma > delta > beta. Exploration of down-stream growth regulators revealed that loss of beta and gamma, but not delta, families was associated with markedly reduced basal ras and protein kinase C activity. Moreover, suppression of each of the three PLC families caused remarkably reduced basal and stimulated MAP kinase activities. Interestingly, cellular levels of PIP2 were increased and dramatically correlated with growth inhibition rate in the clones with suppressed PLC activity, suggesting that PIP2 itself can serve as a second messenger of cell growth regulation.

• Katan M, Williams RL
• Phosphoinositide-specific phospholipase C: structural basis for catalysis and regulatory interactions.
• Semin Cell Dev Biol. 1997; 8: 287-296
• Display abstract

• Phosphoinositide-specific phospholipase C (PI-PLC) isozymes have an important role in cellular responses to a variety of extracellular signals. Recently, the three-dimensional structures of their isolated domains and of the multidomain core, common to all PI-PLCs, have been solved. This provided an insight into the domain organization of PI-PLCs and, together with the structure-function analysis, contributed towards an understanding of the molecular mechanisms of catalysis and regulation.

• Kamat A, Carpenter G
• Phospholipase C-gamma1: regulation of enzyme function and role in growth factor-dependent signal transduction.
• Cytokine Growth Factor Rev. 1997; 8: 109-17
• Display abstract

• Phospholipase C(gamma)1 (PLC-gamma1), a tyrosine kinase substrate, is a multi-domain molecule that modulates the intracellular levels of the second messenger molecules: Ca2+ and diacylglycerol. Although a wide variety of growth factor receptor tyrosine kinases phosphorylate and activate PLC-gamma1, the biological role and necessity of this signal transduction element in mitogenesis has remained unclear. Recent results, however, point to a more essential role than was suggested by initial studies. Also, biochemical studies have indicated a putative means for the intramolecular repression of PLC-gamma1 activity and provide a means for interpreting activation signals through a derepression mechanism.

• Essen LO, Perisic O, Lynch DE, Katan M, Williams RL
• A ternary metal binding site in the C2 domain of phosphoinositide-specific phospholipase C-delta1.
• Biochemistry. 1997; 36: 2753-62
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• We have determined the crystal structures of complexes of phosphoinositide-specific phospholipase C-delta1 from rat with calcium, barium, and lanthanum at 2.5-2.6 A resolution. Binding of these metal ions is observed in the active site of the catalytic TIM barrel and in the calcium binding region (CBR) of the C2 domain. The C2 domain of PLC-delta1 is a circularly permuted topological variant (P-variant) of the synaptotagmin I C2A domain (S-variant). On the basis of sequence analysis, we propose that both the S-variant and P-variant topologies are present among other C2 domains. Multiple adjacent binding sites in the C2 domain were observed for calcium and the other metal/enzyme complexes. The maximum number of binding sites observed was for the calcium analogue lanthanum. This complex shows an array-like binding of three lanthanum ions (sites I-III) in a crevice on one end of the C2 beta-sandwich. Residues involved in metal binding are contained in three loops, CBR1, CBR2, and CBR3. Sites I and II are maintained in the calcium and barium complexes, whereas sites II and III coincide with a binary calcium binding site in the C2A domain of synaptotagmin I. Several conformers for CBR1 are observed. The conformation of CBR1 does not appear to be strictly dependent on metal binding; however, metal binding may stabilize certain conformers. No significant structural changes are observed for CBR2 or CBR3. The surface of this ternary binding site provides a cluster of freely accessible liganding positions for putative phospholipid ligands of the C2 domain. It may be that the ternary metal binding site is also a feature of calcium-dependent phospholipid binding in solution. A ternary metal binding site might be a conserved feature among C2 domains that contain the critical calcium ligands in their CBR's. The high cooperativity of calcium-mediated lipid binding by C2 domains described previously is explained by this novel type of calcium binding site.

• Wu Y, Zhou C, Roberts MF
• Stereocontrolled syntheses of water-soluble inhibitors of phosphatidylinositol-specific phospholipase C: inhibition enhanced by an interface.
• Biochemistry. 1997; 36: 356-63
• Display abstract

• Three inositol 1,2-(cyclic)-phosphate analogs, inositol cyclic phosphonates with different stereochemistry at the C-2 position of the inositol ring, have been synthesized as water-soluble inhibitors of phosphatidylinositol-specific phospholipase C (PI-PLC). Their inhibition of both phosphotransferase and cyclic phosphodiesterase activities has been studied in the absence and presence of an interface. Key results include the following. (i) Only the analog with the same stereochemistry at the C-2 position of the inositol ring as the natural substrate, myo-inositol 1,2-(cyclic)-phosphate (cIP), exhibits effective inhibition of PI-PLC. (ii) The inhibition of the PI-PLC cyclic phosphodiesterase activity by this cIP analog is enhanced by the presence of an interface (Triton X-100 or diC7PC micelles). This is the first observation of detergent enhancing the effectiveness of a water-soluble inhibitor competing with a water-soluble substrate. (iii) For the cyclic phosphodiesterase activity measured in the presence of 8 mM of the best (e.g., most activating) interface, diC7PC, myo-inositol 1,2-(cyclic)-2-methylenephosphonate (cICH2P) was shown to be a competitive inhibitor with a Ki of 12.3 mM. (iv) The IC50 obtained for the same compound inhibiting the PI-PLC hydrolysis of PI dispersed in DiC7PC micelles was consistent with a Ki approximately 10 mM for the phosphotransferase activity. The similarity of Ki for both PI and cIP processing by PI-PLC suggests both reactions occur at the same site on the enzyme.

• Hendrickson HS, Giles AN, Vos SE
• Activity of phosphatidylinositol-specific phospholipase C from Bacillus cereus with thiophosphate analogs of dimyristoylphosphatidylinositol.
• Chem Phys Lipids. 1997; 89: 45-53
• Display abstract

• Phosphatidylinositol-specific phospholipase C (PI-PLC) was studied with sonicated dispersions of a thiophosphate analog of phosphatidylinositol, 1, 2-dimyristoyloxypropane-3-thiophospho(1D-1-myo-inositol) (D-thio-DMPI). Kinetic parameters were derived from the rate as a function of bulk lipid concentration at constant saturating surface concentration of substrate (case I), and as a function of surface concentration of substrate at a constant saturating bulk concentration of lipid (case II). The substrate, D-thio-DMPI, was diluted with L-thio-DMPI or dimyristoyl phosphatidylmethanol (DMPM). In the presence of L-thio-DMPI, values for Vmax = 133 mumol min-1 mg-1, Ks' (the apparent dissociation constant for the enzyme-interface complex) = 0.097 mM, and Km* (the apparent interfacial Michaelis constant) = 0.22 mol fraction were obtained. DMPM caused enzyme inhibition in case I but no inhibition in case II. L-Thio-DMPI is an ideal neutral diluent with which to study the kinetics of PI-PLC.

• Zhou C, Wu Y, Roberts MF
• Activation of phosphatidylinositol-specific phospholipase C toward inositol 1,2-(cyclic)-phosphate.
• Biochemistry. 1997; 36: 347-55
• Display abstract

• Phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus thuringiensis catalyzes the hydrolysis of phosphatidylinositol (PI) in discrete steps: (i) an intramolecular phosphotransferase reaction to form inositol 1,2-(cyclic)-phosphate (cIP), followed by (ii) a cyclic phosphodiesterase activity that converts cIP to inositol 1-phosphate. Water-soluble cIP was used as the substrate to study the cyclic phosphodiesterase activity and interfacial behavior of PI-PLC. Different detergent micelles and phospholipid vesicles were used to examine if "interfacial activation" of the enzyme could occur toward a soluble substrate. Almost all detergents examined activated the enzyme at least 2-fold, with PC species yielding the largest increases in PI-PLC specific activity. Kinetic parameters were measured in the absence and presence of several representative detergents (e.g., Triton X-100 and diheptanoylphosphatidylcholine (diC7PC)). Gel filtration experiments showed that, under these conditions, the cIP did not partition to any measurable extent with these detergent micelles. The concentration at which half the maximum activation was observed occurred near the detergent CMC. Both Km and Vmax were altered by the presence of a surface: Km decreased to different degrees depending on the detergent, while Vmax increased substantially. The Km for cIP was 90 mM without detergent and decreased to 29 mM with diC7PC micelles added; Vmax increased almost 7-fold in the presence of diC7PC micelles. The enzyme efficiency (Vmax/Km) in the presence of diC7PC increased more than 21-fold, but it was still 20-fold lower than initial phosphotransferase activity for monomeric dihexanoylphosphatidylinositol. The poor efficiency of the cyclic phosphodiesterase activity is largely due to substrate binding affinity. The dependence of rate on substrate concentration exhibits cooperative behavior, especially without detergent. This cooperativity is discussed in terms of protein aggregation and ligand binding sites on the enzyme.

• Wu Y, Roberts MF
• Phosphatidylinositol-specific phospholipase C cyclic phosphodiesterase activity depends on solvent polarity.
• Biochemistry. 1997; 36: 8514-21
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• Large enhancements (maximum of 82-fold in terms of enzyme efficiency, Vmax/Km) of bacterial PI-PLC cyclic phosphodiesterase activity were observed in the presence of organic solvents miscible in water (dimethyl sulfoxide, dimethylformamide, and 2-propanol). In general, organic solvents lowered the Km for myo-inositol 1,2-cyclic phosphate (cIP) and increased Vmax substantially. This kinetic effect was similar to that obtained with phosphatidylcholine micelles and bilayers in an aqueous assay system for cyclic inositol phosphate hydrolysis [Zhou, C., et al. (1997) Biochemistry 36, 347-355]. Solvent properties were examined to determine which ones correlated with the activation of PI-PLC toward cIP in each solvent. Activation correlated best with the solvent polarity as measured by ET(30); no significant correlation was observed with solution surface tension, the bulk dielectric constant (epsilon), 1/epsilon (a measure of the strength of charge interactions), or the Hildebrand solubility parameter. The sigmoidal curve of the enzyme activity versus solvent polarity was consistent with the solvent promoting a transition in the enzyme from a low-activity to a high-activity form. Possible candidates for this change, including enzyme dimerization, helix B/loop stabilization, and dehydration of the active site, are discussed.

• Chung JK et al.
• Synaptojanin inhibition of phospholipase D activity by hydrolysis of phosphatidylinositol 4,5-bisphosphate.
• J Biol Chem. 1997; 272: 15980-5
• Display abstract

• A 150-kDa protein that inhibits phospholipase D (PLD) activity stimulated by ADP-ribosylation factor and phosphatidylinositol 4, 5-bisphosphate (PI(4,5)P2) was previously purified from rat brain. The sequences of peptides derived from the purified PLD inhibitor now identify it as synaptojanin, a nerve terminal protein that has been implicated in the endocytosis of fused synaptic vesicles and shown to be a member of the inositol polyphosphate 5-phosphatase family. Further characterization of the enzymatic properties of synaptojanin now shows that it hydrolyzes only the 5-phosphate from inositol 1,4,5-trisphosphate (I(1,4,5)P3) and that it does not catalyze the dephosphorylation of either I(1,3,4)P3 or inositol 1, 4-bisphosphate. However, synaptojanin hydrolyzes both the 4- and 5-phosphates of PI(4,5)P2 and the 4-phosphate of phosphatidylinositol 4-phosphate, converting both compounds to phosphatidylinositol. Magnesium is required for the hydrolysis of I(1,4,5)P3, but not for that of phosphoinositides, by synaptojanin. The inhibition of PLD by synaptojanin is attributable to its ability to hydrolyze PI(4,5)P2. Synaptojanin did not inhibit PLD in the absence of PI(4,5)P2, and the extent of PLD inhibition was related to the extent of PI(4,5)P2 hydrolysis in substrate vesicles. It has been proposed that the biosynthesis of PI(4,5)P2 and the activation of PLD by ADP-ribosylation factor constitute a positive loop to increase rapidly the concentrations of PI(4,5)P2 and phosphatidic acid (PA) during membrane vesiculation. The PA thus produced, probably together with PI(4,5)P2, facilitates vesicle coat assembly. The hydrolysis of PI(4,5)P2, and consequent inhibition of PLD, by synaptojanin might therefore constitute a mechanism to halt the positive loop connecting PI(4,5)P2 and PA during the endocytotic cycle of synaptic vesicles and serve as a signal for uncoating.

• Van Dijken P, Bergsma JC, Van Haastert PJ
• Phospholipase-C-independent inositol 1,4,5-trisphosphate formation in Dictyostelium cells. Activation of a plasma-membrane-bound phosphatase by receptor-stimulated Ca2+ influx.
• Eur J Biochem. 1997; 244: 113-9
• Display abstract

• Dictyostelium cells have enzyme activities that generate the inositol polyphosphate Ins(1,4,5)P3 from Ins(1,3,4,5,6)P5 via the intermediates Ins(1,3,4,5)P4 and Ins(1,4,5,6)P4. These enzyme activities could explain why cells with a deletion of the single phospholipase C gene (plc- cells) possess nearly normal Ins(1,4,5)P3 levels. In this study the regulation and the subcellular localization of the enzyme activities was investigated. The enzyme activities performing the different reaction steps from Ins(1,3,4,5,6)P5 to Ins(1,4,5)P3 are probably due to a single enzyme. Indications for this are the previously shown similar Ca2+ dependencies of the various reaction steps. Furthermore, the activities mediating the complete conversion of Ins(1,3,4,5,6)P5 to Ins(1,4,5)P3 co-purify after subcellular fractionation, solubilization, and chromatography of the proteins. Subcellular fractionation studies demonstrate that the enzyme is localized mainly at the inner face of the plasma membrane. The enzyme activity could not be stimulated in vitro by guanosine 5'-(3-thio)triphosphate, a procedure known to activate G-protein-coupled enzymes in Dictyostelium. Still, in plc- cells the level of Ins(1,4,5)P3 was increased significantly after stimulation with high concentrations of the extracellular ligand cAMP. This stimulation is most likely due to the influx of Ca2+ because no increase of Ins(1,4,5)P3 could be detected in the absence of extracellular Ca2+. The results demonstrate the existence of a new receptor-controlled route for the formation of Ins(1,4,5)P3 that is independent of phospholipase C.

• Wang JP, Hsu MF, Kuo SC
• Inhibition by abruquinone A of phosphoinositide-specific phospholipase C activation in rat neutrophils.
• Eur J Pharmacol. 1997; 319: 131-6
• Display abstract

• In rat neutrophils, formyl-Met-Leu-Phe (fMLP)-induced phosphate formation was inhibited by abruquinone A (IC50 value about 32.7 +/- 6.4 microM) as well as by a putative phospholipase C inhibitor, [6-[[17 beta-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole- 2,5-dione (U73122) (IC50 value about 11.3 +/- 1.2 microM). The reduction in inositol phosphate levels appeared to reflect inhibition of phospholipase C activity because the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) catalyzed by a soluble fraction from neutrophils was also inhibited by abruquinone A (IC50 value about 31.4 +/- 5.6 microM) over the same range of concentrations. Although abruquinone A alone induced Ca2+ and Mn2+ influx into neutrophils in Ca(2+)-containing medium, abruquinone A, like U73122, inhibited Ca2+ release (IC50 value about 23.5 +/- 0.5 microM) from internal stores in Ca(2+)-free medium. These results indicate that abruquinone A inhibits the activity of phosphoinositide-specific phospholipase C in neutrophils.

• Haase I, Liesegang C, Binting S, Henz BM, Rosenbach T
• Phospholipase C-mediated signaling is altered during HaCaT cell proliferation and differentiation.
• J Invest Dermatol. 1997; 108: 748-52
• Display abstract

• To elucidate the signaling mechanisms associated with keratinocyte differentiation, we studied in vitro phospholipase C-mediated signal transduction, which results in the generation of inositol phosphates, comparing proliferating versus differentiated HaCaT cells, a human keratinocyte line. Bradykinin- or A23187-induced formation of inositol 1,4,5-trisphosphate, inositol 1,4-bisphosphate, and inositol monophosphates, as determined by anion exchange high performance liquid chromatography, were found to be highest in the early logarithmic growth phase of the cells. In more highly differentiated HaCaT cells, which expressed maximal amounts of the differentiation marker involucrin, inositol phosphate formation was reduced to about one third of that in proliferating cells. Thin layer chromatography of membrane phosphatidylinositol phosphates revealed that this reduction was associated with a steady decrease in phospholipase C substrates. Immunoblot analysis of phospholipase C isozymes, however, and of expression of Gq alpha, the G protein subunit that activates phospholipase C beta, revealed no decrease during the differentiation phase. The results suggest that the inositol-phospholipid signal transduction pathway is involved in keratinocyte proliferation and in the induction of differentiation, with attenuated signal transduction activity via phospholipase C-coupled receptors in more differentiated keratinocytes.

• Cockcroft S
• Phosphatidylinositol transfer proteins: requirements in phospholipase C signaling and in regulated exocytosis.
• FEBS Lett. 1997; 410: 44-8
• Display abstract

• Phosphatidylinositol transfer proteins (PITP) are abundant cytosolic proteins originally identified because of their ability to act in vitro as specific transporters of phosphatidylinositol or phosphatidylcholine between membranes. However, the cellular function of mammalian PITP has remained enigmatic till recently. Due to the development of reconstitution assays in cytosol-depleted cells, PITP was found to be an essential component for phospholipase C-mediated hydrolysis of PIP2 and for regulated exocytosis. The exact mechanism how PITP exerts its effects is not known but the PI binding/transfer activity of PITP can partly explain its cellular function. PITP would enable the local synthesis of PIP2 by delivering PI to specialized signaling sites.

• Homma MK, Yamasaki M, Ohmi S, Homma Y
• Inhibition of phosphoinositide hydrolysis and cell growth of Swiss 3T3 cells by myristoylated phospholipase C inhibitor peptides.
• J Biochem (Tokyo). 1997; 122: 738-42
• Display abstract

• It has been demonstrated that the phospholipase C-gamma (PLC-gamma) molecule contains within it a phospholipase C inhibitor (PCI) region and that synthetic peptides based on the sequence of this region (PCI peptides) suppress the enzymatic activity of PLC isoforms [Y. Homma and T. Takenawa (1992) J. Biol. Chem. 267, 21884-21889]. In order to improve the permeability of the plasma membrane to PCI peptides, we synthesized myristoylated PCI peptides, myr-GLYRKAMRLRYPV [myr-PCI(Y)] and myr-GLFRKAMRLRFPV [myr-PCI(F)], which are identical except for the replacement of the two tyrosine residues in myr-PCI(Y) by phenylalanines in myr-PCI(F), and examined their inhibitory activity on PLC enzymes in vitro and in vivo. This fatty acid modification potentiated the inhibitory activity of the original PCI peptides and both myr-PCI(Y) and myr-PCI(F) suppressed the PIP2-hydrolyzing activity of purified PLC isoforms in vitro. The Ki values of myr-PCI(Y) and myr-PCI(F) for purified PLC-gamma1 were 3.5 and 55 microM, respectively. Myr-PCI(Y) at concentrations in the sub-micromolar range significantly suppressed IP3 formation induced by EGF, PDGF, bombesin, or serum in Swiss 3T3 cells. Furthermore, myr-PCI(Y) also strongly inhibited cell proliferation induced by these stimuli. The inhibitory effect on IP3 formation and proliferation of myr-PCI(F) was much less potent than that of myr-PCI(Y). These results suggest that myristoylated PCI peptides could be applied to living cells as specific inhibitors of PLC signaling pathways and that PLC pathways are at least in part required for growth in Swiss 3T3 cells.

• Lim SJ, Kim CK
• Homogeneous liposome immunoassay for insulin using phospholipase C from Clostridium perfringens.
• Anal Biochem. 1997; 247: 89-95
• Display abstract

• A new homogeneous liposome immunoassay system was developed in which analyte-phospholipase C conjugates are used instead of complement or melittin. This system was applied for the determination of insulin. Liposomes incorporated with calcein as a marker were prepared by the reverse-phase evaporation method. The lysis of liposomes was determined by measuring the fluorescence intensity of calcein released from liposomes and it was increased with increasing concentration of phospholipase C. Insulin was conjugated to phospholipase C by a three-step procedure with hetero-bifunctional crosslinking reagents, succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate and 3-(2-pyridyldithio)propionic acid N-hydroxysuccinimide ester. The lytic activity of phospholipase C was not affected by the reaction for conjugation. Both p-nitrophenylphosphatidylcholine hydrolytic activity and liposome lytic activity of insulin-phospholipase C conjugate were inhibited in the presence of insulin antiserum. However, lower concentration of conjugate and shorter incubation time were required when liposomes were used in the inhibition study. The antibody inhibition of conjugate-induced lysis could be reversed by addition of a competing amount of free insulin. The standard calibration curve was obtained in the range between 4 and 130 microIU/ml (r = 0.994). The detection limit (8 microIU/ml) was comparable with those of conventional heterogeneous enzyme immunoassays. This new assay approach offers a simple, sensitive, and inexpensive testing procedure for determining ultratrace amounts of bioactive substances.

• Grobler JA, Hurley JH
• Similarity between C2 domain jaws and immunoglobulin CDRs.
• Nat Struct Biol. 1997; 4: 261-2

• Zhou C, Qian X, Roberts MF
• Allosteric activation of phosphatidylinositol-specific phospholipase C: specific phospholipid binding anchors the enzyme to the interface.
• Biochemistry. 1997; 36: 10089-97
• Display abstract

• Phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus thuringiensis exhibits 'interfacial activation' toward the water-soluble substrate myo-inositol 1,2-(cyclic)phosphate [Zhou et al. (1997) Biochemistry 36, 347-355]. The activation of PI-PLC enzyme is optimal with PC or PE interfaces. NMR experiments (TRNOE and 31P line width analyses) were carried out to investigate the interaction of PI-PLC with activator amphiphiles. These studies showed that the enzyme had high affinity for phosphatidylcholine (or PE) molecules with dissociation constants of 0.5 and 0.3 mM for diC6PC and diC7PC, respectively. TRNOE cross-peaks of bound PC were confirmed to represent intramolecular relaxation pathways using partially perdeuterated PC molecules consistent with a single molecule binding tightly. The large activation by a PC interface can be explained by a single PC molecule binding specifically to PI-PLC and anchoring the enzyme-lipid complex to the interface. Other interfaces, such as micellar diC8PS, can activate PI-PLC about 2-3-fold; however, the monomers of these detergents showed little affinity for the enzyme as measured by TRNOE or 31P NMR line widths. The 3.6-fold activation produced by polymerized vesicles of 1,2-bis[12-(lipoyloxy)dodecanoyl]-sn-glycero-3-phosphocholine (compared to the 15-fold activation generated by nonpolymerized PC vesicles) was comparable to the nonspecific activation of other detergents. This confirmed that single-PC molecule binding was allosteric and anchored the enzyme in the interface. The conformation of interfacially activated enzyme is discussed in term of the stabilization of a critical surface loop and helix B observed with weak intensity in the X-ray crystal structure.

• Matecki A, Pawelczyk T
• Regulation of phospholipase C delta1 by sphingosine.
• Biochim Biophys Acta. 1997; 1325: 287-96
• Display abstract

• Sphingosine, which is on the pathway of sphingomyelin degradation, activates phospholipase C (PLC) delta1 moderately. In the liposome assay effect of sphingosine on PLC delta1 activity depends on KCl concentration. Stimulation of PLC delta1 by sphingosine increased as the KCl concentration is increased from 0 to 100 mM, and then diminished with the increasing KCl. In the liposome assay sphingosine diminishes inhibition of PLC delta1 by sphingomyelin. To determine the domain of PLC delta1 which interacts with sphingosine active proteolytic fragments of PLC delta1 were generated by trypsin digestion of the native enzyme. Sphingosine affects the activity of PLC delta1 fragment which lacked the amino-terminal domain (first 60 amino acids) but not the active fragment that has cleaved the domain spanning the X and Y region of PLC delta1. These observations indicate that for interaction of sphingosine with PLC delta1 intact domain that span regions of conservation, designated as X and Y is necessary. When the activity of PLC delta1 was assayed with PIP2 in the erythrocyte membrane as substrate, sphingosine strongly inhibited PLC delta1. The other homolog of sphingosine 4-hydroxysphinganine (phytosphingosine) inhibited PLC delta1 to much lesser extent. The activity of PLC delta1 was inhibited by 68% and 22% in the presence of 20 microM sphingosine and phytosphingosine, respectively. This inhibition was completely abolished by deoxycholate at a concentration of 1.5 mM. These observations suggest that sphingosine may regulate activity of PLC delta1 in the cell.

• Itami C, Kimura Y, Taguchi R, Ikezawa H, Nakabayashi T
• Growth inhibition, morphological change, and ectoenzyme release of LLC-PK1 cells by phosphatidylinositol-specific phospholipase C of Bacillus thuringiensis.
• Biosci Biotechnol Biochem. 1997; 61: 776-81
• Display abstract

• Phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus thuringiensis added to a culture of LLC-PK1 cells inhibited cell growth by 40%. In contrast with normal cells, the cells cultured in the presence of PI-PLC showed needle-like appendages which seemed to have been formed due to portions of the cell remaining adhered to the culture dish as the cell shrank. When LLC-PK1 cells were treated with PI-PLC, significant amounts of alkaline phosphatase and alkaline phosphodiesterase I were released specifically from the apical surface of the LLC-PK1 cells. Furthermore, PI-PLC treatment caused a delay of enzyme production and dome formation. These data indicate that glycosyl-phosphatidylinositol (GPI)-anchored proteins on the surface of LLC-PK1 cells are important in cell growth and differentiation. Also, the combined use of LLC-PK1 cells and PI-PLC of B. thuringiensis is effective for investigating the function of GPI-anchor proteins.

• Igwe OJ, Filla MB
• Aging-related regulation of myo-inositol 1,4,5-trisphosphate signal transduction pathway in the rat striatum.
• Brain Res Mol Brain Res. 1997; 46: 39-53
• Display abstract

• To determine the effects of the aging process on the regulation of phosphoinositide signal transduction pathway, inositol 1,4,5-trisphosphate and inositol 1,4,5-trisphosphate receptor-associated parameters were examined in the striatum of brains removed from young (3 months), adult (12 months) and senescent (25 months) male Fischer 344 rats. Inositol 1,4,5-trisphosphate content was significantly increased (P < or = 0.01) at 25 months of age compared to 3 and 12 months. No age-related differences in phosphatidylinositol 4,5-bisphosphate hydrolysis were found in striatal slices after stimulation with trans-(1S,3R)-1-aminocyclopentane-1,3-dicarboxylate, a metabotropic glutamatergic receptor agonist. Phosphatidylinositol 4,5-bisphosphate hydrolysis following stimulation with (R,S)-alpha-amino-3-hydroxyl-5-methyl-4-isoxazolepropionic acid, a glutamatergic/quisqualate agonist, showed a significantly increased accumulation of net [3H]inositol 1,4,5-trisphosphate in senescent striatum whereas the muscarinic cholinergic agonist carbachol induced highest response in the young striatum. In each case, agonist-stimulated response was significantly reduced in the presence of the receptor-associated antagonist. The density of inositol 1,4,5-trisphosphate receptor in the particulate membranes derived from 12- and 25-month-old rats was decreased (P < 0.01) compared to that from young rats. Binding affinity of inositol 1,4,5-trisphosphate receptor for [3H]inositol 1,4,5-trisphosphate was increased (P = 0.05) only at 25 months of age when compared with 3 months of age. Incubation of partially purified inositol 1,4,5-trisphosphate receptor with striatal cytosol in the presence of Ca2+ showed an age-dependent susceptibility to proteolytic degradation of this receptor that was completely inhibited by calpain I inhibitor peptide. Paradoxically, the quantity of inositol 1,4,5-trisphosphate receptor mRNA-encoding transcripts was increased (P < or = 0.01) at 25 months of age, suggesting an age-dependent change in either transcriptional rate, stability or processing of inositol 1,4,5-trisphosphate receptor mRNAs in the striatum. The activity of inositol 1,4,5-trisphosphate3-kinase decreased (P < or = 0.01) with age whereas the activity of soluble inositol 1,4,5-trisphosphate 5-phosphatase was highest at 3 months but significantly decreased at 12 months of age. However, the activity of inositol 1,4,5-trisphosphate 5-phosphatase remained unchanged between 12 and 25 months of age, suggesting possible developmental modulation of the activity of the enzyme. Taken together with the established 'cross-talk' between signal transduction systems, the present data suggest that molecular/cellular changes in striatal inositol 1,4,5-trisphosphate/Ca2+ signal transduction pathway along with neuronal cell loss may contribute to aging-related decrease in striatal functioning.

• Le Mellay V, Grosse B, Lieberherr M
• Phospholipase C beta and membrane action of calcitriol and estradiol.
• J Biol Chem. 1997; 272: 11902-7
• Display abstract

• We have shown that estrogens and calcitriol, the hormonally active form of vitamin D, increase the concentration of intracellular calcium ([Ca2+]i) within 5 s by mobilizing calcium from the endoplasmic reticulum and the formation of inositol 1,4, 5-trisphosphate and diacylglycerol. Because the activation of effectors as phospholipase C (PLC) coupled to G-proteins is the early event in the signal transduction pathway leading to the inositol 1,4,5-trisphosphate formation and to [Ca2+]i increase, we described different PLC isoforms (beta1, beta2, gamma1, and gamma2, but not beta4) in female rat osteoblasts using Western immunoblotting. The data showed that phospholipase C beta was involved in the mobilization of Ca2+ from the endoplasmic reticulum of Fura-2-loaded confluent osteoblasts by calcitriol and 17beta estradiol, and PLC gamma was ineffective. The data also showed that only a PLC beta1 linked to a Pertussis toxin-insensitive G-protein and a PLC beta2 coupled to a Pertussis toxin-sensitive G-protein are involved in the effects of calcitriol and 17beta estradiol on the mobilization of Ca2+ from intracellular Ca2+ stores. In conclusion, these results may be an important step toward understanding membrane effects of these steroids and may be an additional argument in favor of membrane receptors to steroid hormones.

• Malm D, Lindskog S, Ahren B, Florholmen J
• Galanin exerts dual action on inositol-specific phospholipase C activity in isolated pancreatic islets.
• Endocr J. 1997; 44: 283-8
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• The intracellular mechanism whereby the neuropeptide galanin inhibits insulin secretion is not establish, since the peptide affects several signal pathways, including intracellular messengers such as calcium and cyclic AMP. In this study, we have assessed the effect of galanin on the inositol-specific phospholipase C (iPLC) activity in isolated rat pancreatic islets. The iPLC activity was measured as the generation of inositol 1,4,5-trisphosphate and its metabolite inositol 1,3,4-trisphosphate from the hydrolysis of polyphosphoinositides. Inositol phosphates were measured by anion-exchange fast protein liquid chromatography (FPLC) analysis of extracts from islets prelabelled with myo-3H-inositol. Galanin (1 to 100 nM) significantly increased the glucose-induced (12 mM) accumulation of inositol 1,4,5-trisphosphate after 2 min, but this stimulation of iPLC activity was followed by a significant suppression after 15 min. In the absence of extracellular calcium, both the stimulatory and inhibitory effects of galanin on the iPLC activity vanished. We therefore conclude that galanin initially stimulates iPLC in a calcium-dependent manner, followed by a secondary inhibitory effect. The secondary inhibition of iPLC activity might contribute to the insulinostatic action of the neuropeptide.

• Pawelczyk T, Matecki A
• Structural requirements of phospholipase C delta1 for regulation by spermine, sphingosine and sphingomyelin.
• Eur J Biochem. 1997; 248: 459-65
• Display abstract

• We studied the relationship between sphingomyelin, calcium, spermine and sphingosine in regulation of phospholipase C (PLC) delta1 activity. Inhibition of PLC delta1 by sphingomyelin was promoted by spermine and Ca2+ and was partially abolished by sphingosine. The effect of sphingosine and spermine entirely depended on Ca2+. In the absence of Ca2+, no effect of these substances on PLC delta1 activity was observed. Using deletion mutants and active fragments of PLC delta1 generated by limited proteolysis, we have studied the structural requirements of the enzyme for regulation by these compounds. The deletion mutant of PLC delta1 lacking the first 58 amino acids and the mutant lacking the entire pleckstrin homology (PH) domain were fully active in the detergent assay, and their activities were affected by spermine, sphingosine, Ca2+ and sphingomyelin to the same extent as the native enzyme. The limited proteolysis of PLC delta1 generated two fragments of 40 kDa and 30 kDa, which formed a stable active complex. The relationship between Ca2+ concentration and enzymatic activity was almost identical for the native PLC delta1 and the proteolytic complex. The activity of the proteolytic complex formed by the 40 kDa and 30 kDa peptides was not affected by spermine and sphingosine. Sphingomyelin inhibited the complex slightly less than the native PLC delta1, and this inhibition was not promoted by spermine. These observations suggest that for activation of PLC delta1 by spermine and sphingosine, the region spanning domains of high conservation, named X and Y, must be intact. In contrast, the PH domain and the intact spanning region of the X and Y domains are not essential for inhibition of PLC delta1 by sphingomyelin.

• Liu T, Ryan M, Dahlquist FW, Griffith OH
• Determination of pKa values of the histidine side chains of phosphatidylinositol-specific phospholipase C from Bacillus cereus by NMR spectroscopy and site-directed mutagenesis.
• Protein Sci. 1997; 6: 1937-44
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• Two active site histidine residues have been implicated in the catalysis of phosphatidylinositol-specific phospholipase C (PI-PLC). In this report, we present the first study of the pKa values of histidines of a PI-PLC. All six histidines of Bacillus cereus PI-PLC were studied by 2D NMR spectroscopy and site-directed mutagenesis. The protein was selectively labeled with 13C epsilon 1-histidine. A series of 1H-13C HSQC NMR spectra were acquired over a pH range of 4.0-9.0. Five of the six histidines have been individually substituted with alanine to aid the resonance assignments in the NMR spectra. Overall, the remaining histidines in the mutants show little chemical shift changes in the 1H-13C HSQC spectra, indicating that the alanine substitution has no effect on the tertiary structure of the protein. H32A and H82A mutants are inactive enzymes, while H92A and H61A are fully active, and H81A retains about 15% of the wild-type activity. The active site histidines, His32 and His82, display pKa values of 7.6 and 6.9, respectively. His92 and His227 exhibit pKa values of 5.4 and 6.9. His61 and His81 do not titrate over the pH range studied. These values are consistent with the crystal structure data, which shows that His92 and His227 are on the surface of the protein, whereas His61 and His81 are buried. The pKa value of 6.9 corroborates the hypothesis of His82 acting as a general acid in the catalysis. His32 is essential to enzyme activity, but its putative role as the general base is in question due to its relatively high pKa.

• Gassler CS, Ryan M, Liu T, Griffith OH, Heinz DW
• Probing the roles of active site residues in phosphatidylinositol-specific phospholipase C from Bacillus cereus by site-directed mutagenesis.
• Biochemistry. 1997; 36: 12802-13
• Display abstract

• The role of amino acid residues located in the active site pocket of phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus cereus[Heinz, D. W., Ryan, M., Bullock, T., & Griffith, O. H. (1995) EMBO J. 14, 3855-3863] was investigated by site-directed mutagenesis, kinetics, and crystal structure analysis. Twelve residues involved in catalysis and substrate binding (His32, Arg69, His82, Gly83, Lys115, Glu117, Arg163, Trp178, Asp180, Asp198, Tyr200, and Asp274) were individually replaced by 1-3 other amino acids, resulting in a total number of 21 mutants. Replacements in the mutants H32A, H32L, R69A, R69E, R69K, H82A, H82L, E117K, R163I, D198A, D198E, D198S, Y200S, and D274S caused essentially complete inactivation of the enzyme. The remaining mutants (G83S, K115E, R163K, W178Y, D180S, Y200F, and D274N) exhibited reduced activities up to 57% when compared with wild-type PI-PLC. Crystal structures determined at a resolution ranging from 2.0 to 2.7 A for six mutants (H32A, H32L, R163K, D198E, D274N, and D274S) showed that significant changes were confined to the site of the respective mutation without perturbation of the rest of the structure. Only in mutant D198E do the side chains of two neighboring arginine residues move across the inositol binding pocket toward the newly introduced glutamic acid. An analysis of these structure-function relationships provides new insight into the catalytic mechanism, and suggests a molecular explanation of some of the substrate stereospecificity and inhibitor binding data available for this enzyme.

• Slaaby R, Knott T, Davey J
• Characterisation of a phospholipase C delta from Schizosaccharomyces pombe.
• Biochem Soc Trans. 1997; 25: 225-225

• Schlessinger J
• Phospholipase Cgamma activation and phosphoinositide hydrolysis are essential for embryonal development.
• Proc Natl Acad Sci U S A. 1997; 94: 2798-9

• Ji QS et al.
• Essential role of the tyrosine kinase substrate phospholipase C-gamma1 in mammalian growth and development.
• Proc Natl Acad Sci U S A. 1997; 94: 2999-3003
• Display abstract

• The activation of many tyrosine kinases leads to the phosphorylation and activation of phospholipase C-gamma1 (PLC-gamma1). To examine the biological function of this protein, homologous recombination has been used to selectively disrupt the Plcg1 gene in mice. Homozygous disruption of Plcg1 results in embryonic lethality at approximately embryonic day (E) 9.0. Histological analysis indicates that Plcg1 (-/-) embryos appear normal at E 8.5 but fail to continue normal development and growth beyond E 8.5-E9.0. These results clearly demonstrate that PLC-gamma1 with, by inference, its capacity to mobilize second messenger molecules is an essential signal transducing molecule whose absence is not compensated by other signaling pathways or other genes encoding PLC isozymes.

• Hurley JH, Grobler JA
• Protein kinase C and phospholipase C: bilayer interactions and regulation.
• Curr Opin Struct Biol. 1997; 7: 557-65
• Display abstract

• Protein kinase C and phospholipase C are interfacially active modular enzymes that contain multiple membrane-binding domains. During the past two years, 3D structures and functional data have been reported for the key domains: pleckstrin homology, protein kinase C homology-1 and -2, and the phospholipase C catalytic domain. Roles for membrane bilayer structure and lipid microdomains have become clearly domains has shown how the domains work together to coordinate regulation.

• Wu Y, Perisic O, Williams RL, Katan M, Roberts MF
• Phosphoinositide-specific phospholipase C delta1 activity toward micellar substrates, inositol 1,2-cyclic phosphate, and other water-soluble substrates: a sequential mechanism and allosteric activation.
• Biochemistry. 1997; 36: 11223-33
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• The kinetics of full-length and PH domain truncated cloned PI-PLC delta1 from rat toward soluble substrates [inositol 1, 2-(cyclic)-phosphate (cIP) and glycerophosphoinositol phosphates (GPIPx)] as well as PI in detergent micelles provide the following insights into the mechanism of this enzyme. (i) That cIP is a substrate for the enzyme implies a two-step mechanism for PI hydrolysis [intramolecular phosphotransferase reaction to form cIP followed by cyclic phosphodiesterase activity to form inositol-1-phosphate (I-1-P)]. The dependence of enzyme activity on cIP is sigmoidal, suggesting a transition between less active and more active forms of the enzyme that is affected by substrate. (ii) Interfaces increase the kcat for cIP (but do not affect the cooperativity), and this allosteric activation requires an intact PH domain. (iii) Phosphorylation of the soluble inositol phosphodiesters GPI, GPIP, and GPIP2 enhances PI-PLC delta1 activity by dramatically increasing kcat and decreasing Km. For these phosphodiesters, the substrate saturation curve is no longer sigmoidal but hyperbolic, indicating the phosphorylated substrate can shift the enzyme to the activated form. (iv) Given the kinetic parameters for cIP hydrolysis and the constant ratio of cIP/I-1-P generated during PI hydrolysis, the cIP produced in situ is either released (and not readily rebound since its concentration is well below Km) or attacked by a water molecule for the generation of the acyclic product.

• James SR, Downes CP
• Structural and mechanistic features of phospholipases C: effectors of inositol phospholipid-mediated signal transduction.
• Cell Signal. 1997; 9: 329-36
• Display abstract

• The production of the intracellular second messengers inositol (1,4,5)-trisphosphate (InsP3) and sn 1,2-diacylglycerol (DG) in response to a wide variety of extracellular primary messengers is achieved by an extended family of inositol phospholipid phosphodiesterases termed phospholipases C (PLC, E.C. 3.1.4.11). This family has been the subject of extensive research and it is clear that the different isoenzymes exhibit some common characteristics (e.g., interactions with substrates) and other distinctive features (e.g., modes of regulation). The recent description of the X-ray crystal structure of a mammalian PLC has served to clarify much about the behaviour of the PLCs, emphasising the "modular" structure of these enzymes. The main focus of this review will concern the specific adaptations of PLC molecules which make them efficient lipid-metabolising enzymes. We also describe what is known about how these enzymes interact with their lipid substrates, which will serve as a basis for considering how PLCs may be activated.

• Rukavishnikov AV, Zaikova TO, Griffith OH, Keana JF
• Improved synthesis of myo-inositol 1-(4-nitrophenyl hydrogen phosphate), a chromogenic substrate for phosphatidylinositol-specific phospholipase C.
• Chem Phys Lipids. 1997; 89: 153-7

• Kim D et al.
• Phospholipase C isozymes selectively couple to specific neurotransmitter receptors.
• Nature. 1997; 389: 290-3
• Display abstract

• A variety of extracellular signals are transduced across the cell membrane by the enzyme phosphoinositide-specific phospholipase C-beta (PLC-beta) coupled with guanine-nucleotide-binding G proteins. There are four isoenzymes of PLC-beta, beta1-beta4, but their functions in vivo are not known. Here we investigate the role of PLC-beta1 and PLC-beta4 in the brain by generating null mutations in mice: we found that PLCbeta1-/- mice developed epilepsy and PLCbeta4-/- mice showed ataxia. We determined the molecular basis of these phenotypes and show that PLC-beta1 is involved in signal transduction in the cerebral cortex and hippocampus by coupling predominantly to the muscarinic acetylcholine receptor, whereas PLC-beta4 works through the metabotropic glutamate receptor in the cerebellum, illustrating how PLC-beta isoenzymes are used to generate different functions in the brain.

• Carnall N, Webb H, Carrington M
• Mutagenesis study of the glycosylphosphatidylinositol phospholipase C of Trypanosoma brucei.
• Mol Biochem Parasitol. 1997; 90: 423-32
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• The glycosylphosphatidylinositol phospholipase C (GPI-PLC) from Trypanosoma brucei is particularly effective in hydrolysing the GPI-anchors of some proteins. The enzyme is inhibited by Zn2+ and p-chloromercurylphenylsulphonic acid, both of which can act as sulphydryl reagents, suggesting that a cysteine residue may be important in catalysis. Single cysteine to serine mutants have been produced for all eight cysteines in GPI-PLC; all the mutants were fully active in vitro and were still susceptible to p-chloromercurylphenylsulphonic acid inhibition. In contrast, a single histidine 34 to glutamine mutation totally inactivated GPI-PLC. The histidine was chosen after a sequence alignment with the Bacillus cereus phosphatidylinositol phospholipase C (PI-PLC) suggested a conservation of active site residues, including histidine 34 which is central to the proposed reaction mechanism (Heinz D.W., Ryan M., Bullock T.L., Griffith O.H. EMBO J 1995;14:3855-3863). The results suggest that the GPI-PLC and bacterial PI-PLCs have conserved active sites and that the inhibition of GPI-PLC by sulphydryl reagents can occur through more than one residue.

• James SR, Paterson A, Harden TK, Demel RA, Downes CP
• Dependence of the activity of phospholipase C beta on surface pressure and surface composition in phospholipid monolayers and its implications for their regulation.
• Biochemistry. 1997; 36: 848-55
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• We have examined the influence of surface pressure and phospholipid composition on hydrolysis of phosphatidylinositol (4,5)-bisphosphate (PIP2) by phospholipase C beta 1 (PLC beta 1) and PLC beta 2 in mixed composition phospholipid monolayers. Increasing the monolayer surface pressure from 15 to 36 mN/m reduced the rate at which PIP2 was hydrolyzed by PLC beta 1 and PLC beta 2 by 4-6-fold, although PLC beta 1 was more active than PLC beta 2, even at high surface pressure. Reduced enzyme activity was accompanied by an increase in reaction induction times, suggesting that increasing surface pressure reduced the penetration rate of the enzymes into the monolayer. Quantitation of interfacial enzyme concentration using 35S-labeled PLC beta 1 confirmed that less enzyme was associated with the monolayer at higher pressures. The relationship between PLC activity and substrate concentration was examined at a single surface pressure of 30 mN/m. This relationship was not hyperbolic, and increases in the mole percentage (mol %) of PIP2 in the monolayer resulted in an upwardly-curving increase in PLC activity. Thus, PLC beta 1 activity increased 7-fold and PLC beta 2 activity increased 4-fold when the mol % of PIP2 in the monolayer increased from 17.9% to 29%, increasing further thereafter. Paradoxically, increasing the mol % of PIP2 from 0 to 60% was accompanied by a 3-fold decrease in interfacial enzyme concentrations. Taken together, these data show that the catalytic activity of PLC beta involves some element of penetration of lipid interfaces, and suggest that the organization of the substrate facilitates PLC activity, giving credence to the substrate theory of interfacial activation of phospholipases. We present a hypothesis suggesting that PIP2 molecules coalesce into enriched lateral domains which favor PLC beta activity.

• Hendrickson HS, Banovetz C, Kirsch MJ, Hendrickson EK
• Kinetics of phosphatidylinositol-specific phospholipase C with vesicles of a thiophosphate analogue of phosphatidylinositol.
• Chem Phys Lipids. 1996; 84: 87-92
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• 1,2-Dimyristoyloxypropane-3-thiophospho(1D-1-myo-inositol) (D-thio-DMPI) was synthesized as a substrate for the continuous spectrophotometric assay of phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus cereus. Release of thio-diglyceride is followed by a coupled reaction with 4,4'-dithiopyridine to produce a chromophore, 4-thiopyridine, measured by its absorption at 324 nm. Sonicated vesicles of D-thio-DMPI gave sigmoidal Michaelis-Menten kinetics with PI-PLC as a function of bulk concentration of substrate (Hill plot: Vmax = 132 mumol min-1 mg-1, apparent Km = 0.115 mM, h = 1.8). Addition of dimyristoyl phosphatidylcholine (DMPC) or dimyristoyl phosphatidylmethanol to vesicles of D-thio-DMPI resulted in an initial increase in rate followed by a decrease at higher concentrations of non-substrate lipid. Binding of PI-PLC to vesicles of DMPC with 10 mol% of N-dansyl phosphatidylethanolamine was demonstrated by fluorescence resonance energy transfer from tryptophan in the enzyme to the dansyl lipid probe.

• Raboy V, Gerbasi P
• Genetics of myo-inositol phosphate synthesis and accumulation.
• Subcell Biochem. 1996; 26: 257-85

• Reo NV, Narayanan L, Kling KB, Adinehzadeh M
• Perfluorodecanoic acid, a peroxisome proliferator, activates phospholipase C, inhibits CTP:phosphocholine cytidylyltransferase, and elevates diacylglycerol in rat liver.
• Toxicol Lett. 1996; 86: 1-11
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• Perfluorooctanoic acid (PFOA) and perfluorodecanoic acid (PFDA) are peroxisome proliferators that cause hepatotoxicity in rodents. This study shows that PFDA activates liver phospholipase C (PLC) and inhibits CTP:phosphocholine cytidylyltransferase (CT). PLC cytosolic and microsomal activities were increased 1.4- and 1.7-fold, respectively. CT activates were decreased to 58% (cytosol) and 36% (microsome) of control values. PFDA also caused a threefold increase in liver diacylglycerol (DAG) concentration. PFOA had no effect on the enzyme activities or DAG concentration. Together with previous results, these data suggest that PFDA activates a phosphatidylcholine-specific PLC causing an increase in liver phosphocholine and DAG. These effects are discussed in relation to cellular signalling processes that may provide a mechanism for PFDA-induced hepatotoxicity.

• Glaser M et al.
• Myristoylated alanine-rich C kinase substrate (MARCKS) produces reversible inhibition of phospholipase C by sequestering phosphatidylinositol 4,5-bisphosphate in lateral domains.
• J Biol Chem. 1996; 271: 26187-93
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• The myristoylated alanine-rich protein kinase C substrate (MARCKS) is a major protein kinase C (PKC) substrate in many different cell types. MARCKS is bound to the plasma membrane, and several recent studies suggest that this binding requires both hydrophobic insertion of its myristate chain into the bilayer and electrostatic interaction of its cluster of basic residues with acidic lipids. Phosphorylation of MARCKS by PKC introduces negative charges into the basic cluster, reducing its electrostatic interaction with acidic lipids and producing translocation of MARCKS from membrane to cytoplasm. The present study shows that physiological concentrations of MARCKS (<10 microM) inhibit phospholipase C (PLC)-catalyzed hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) in phospholipid vesicles. A peptide corresponding to the basic cluster, MARCKS(151-175), produces a similar inhibition, which was observed with both PLC-delta1 and -beta1. Direct fluorescence microscopy observations demonstrate that the MARCKS peptide forms lateral domains enriched in the acidic lipids phosphatidylserine and PIP2 but not PLC, which accounts for the observed inhibition of PIP2 hydrolysis. Phosphorylation of MARCKS(151-175) by PKC releases the inhibition and allows PLC to produce a burst of inositol 1,4, 5-trisphosphate and diacylglycerol.

• Venkatakrishnan G, Exton JH
• Identification of determinants in the alpha-subunit of Gq required for phospholipase C activation.
• J Biol Chem. 1996; 271: 5066-72
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• A series of chimeras between a constitutively active mutant of the alpha-subunit of Gq and the alpha-subunit of Gs was constructed to identify the domains in alphaq specifically involved in interaction with its effector phosphoinositide phospholipase C (PLC). Transient expression of the chimeric proteins and measurement of the production of inositol phosphates and cAMP in HEK-293 cells revealed that the Ile217-Lys276 sequence of alphaq contained the PLC interaction sites, whereas the residues for activation of adenylyl cyclase were in the Ile235-Leu294 sequence of alphas. Alanine scanning mutagenesis of the Ile217-Lys276 region of alphaq further identified two clusters of amino acids (Asp243,Asn244,Glu245 and Arg256,Thr257) that were specifically required for interaction with PLC. Comparison of the sequences of alphaq, alphas, and alphat showed that the PLC-interacting residues identified in alphaq are different from the corresponding residues in alphas and alphat that are involved in effector activation. Alignment of the sequences of alphaq and alphat, based on the crystal structure of alphat (Noel, J. P., Hamm, H. E., and Sigler, P. D. (1993) Nature 366, 654-663), indicated that the PLC-activating residues of alphaq are located in alpha-helix 3 and its linker to beta-sheet 4, which are adjacent to a switch region whose conformation changes with activation. It is proposed that the selectivity of alphaq for PLC involves relatively few amino acids, but that the effector may interact with other nonselective sequences in the alpha-subunit.

• Grobler JA, Hurley JH
• Expression, characterization, and crystallization of the catalytic core of rat phosphatidylinositide-specific phospholipase C delta 1.
• Protein Sci. 1996; 5: 680-6
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• Phosphatidylinositide-specific phospholipase Cs (PI-PLCs) catalyze the calcium-dependent hydrolysis of phosphatidylinositides in response to diverse stimuli in higher eukaryotes. Mammalian PI-PLCs contain divergent regulatory regions, but all share three conserved regions: an N-terminal pleckstrin homology (PH) domain, X, and Y. We report the high-level expression and characterization of a recombinant "catalytic core" of rat PI-PLC delta 1 that contains the catalytically essential X and Y regions, but not the PH domain. The expressed protein, PI-PLC delta delta 1-134, is catalytically active versus phosphatidylinositol 4,5-bisphosphate in deoxycholate micelles with a K(m) of 182 microM and a Vmax of 27 mumol/min/mg. PI-PLC delta delta 1-134 is monomeric and monodisperse as judged by dynamic light scattering. Far-UV CD indicates a structure with approximately 35% alpha-helix. A reversible change in the near-UV CD spectrum is observed on addition of calcium, suggesting that calcium can bind PI-PLC delta delta 1-134 in the absence of phospholipid. Triclinic crystals of PI-PLC delta delta 1-134 have been obtained that diffract beyond 2.4 A resolution under cryogenic conditions. Based on Vm = 2.72 Da/A3 and on the self-rotation function, there are two PI-PLC delta delta 1-134 molecules per asymmetric unit that are related to each other by a noncrystallographic axis of approximate twofold symmetry parallel to a.

• Haase I, Czarnetzki BM, Rosenbach T
• Thrombin and melittin activate phospholipase C in human HaCaT keratinocytes.
• Exp Dermatol. 1996; 5: 84-8
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• Following the activation of specific receptors, phospholipase C has been shown to cleave the membrane phospholipid phosphatidylinositol bisphosphate into the 2nd messengers inositol 1,4,5-trisphosphate and diacylglycerol. Both 2nd messengers contribute to the regulation of cellular proliferation. The receptor for bradykinin is coupled to this pathway in keratinocytes, but knowledge about other activators of phospholipase C is limited. Additional mediators and agents were therefore examined regarding their ability to activate phospholipase C in HaCaT keratinocytes. Analysis for 3H-inositol phosphates was performed by anion-exchange HPLC. Thrombin and melittin induced a time- and dose-dependent release of inositol 1,4,5-trisphosphate. Several other mediators examined such as angiotension II, neurotensin, C3a, pituitary adenylate cyclase activating peptide, phenylephrin, and prostaglandin E2, did not induce the formation of inositol phosphates. In view of the mitogenic activity and the increased formation of thrombin after tissue injury, the coupling of the thrombin receptor to phospholipase C in HaCaT keratinocytes suggests a role of this protease in epidermal wound healing.

• Sesca E, Chiara M, Binasco V, Premoselli F, Tessitore L
• Choline feeding depresses the phospholipase C activity in the regenerating liver of female rats.
• Boll Soc Ital Biol Sper. 1996; 72: 325-9
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• The administration of an excess of choline for 3 weeks is able to delay the proliferative response to partial hepatectomy (PH) in female rats. Choline feeding can affect the phospholipid composition of cell membranes and, as a consequence, the transduction of the mitogenic signals. On these bases, we studied the turnover of phosphatidylinositol-4,5-biphosphate (PIP2) in the regenerating liver of female rats. The hydrolysis of PIP2 is catalysed by a specific phospholipase C (PL-C) and it generates the second messenger molecules, namely diacylglycerol and inositol-1,4,5-triphosphate (IP3). Our results showed that the administration of an excess of choline to females was able to reduce the PL-C activity and the membrane IP3 content in the quiescent liver. Both parameters remained lower than controls during liver regeneration, even if they were higher 1 and 2 h after PH in comparison with the quiescent liver, in choline-fed females. These data suggest that the delay in the liver regeneration by choline is due, at least in part, to the alteration in the pathway of PIP2 turnover for the transduction of mitogenic signals.

• Bruch RC
• Phosphoinositide second messengers in olfaction.
• Comp Biochem Physiol B Biochem Mol Biol. 1996; 113: 451-9
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• Olfactory stimuli (odorants) are detected and recognized by binding to receptors belonging to the G-protein-coupled receptor superfamily. The binding of odorants to some receptors stimulates the activity of an odorant-sensitive phospholipase C (PLC) thereby generating the second messengers inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). IP3 plays a key role in membrane depolarization by binding to a receptor that is itself a cation channel. The formation of DAG is expected to stimulate the activity of protein kinase C (PKC). PKC, together with G-protein-coupled receptor kinases, mediates signal termination by phosphorylation of odorant receptors and possibly other substrates. This review summarizes recent evidence regarding the role of phosphoinositide-derived second messengers in the molecular events underlying olfactory signaling. In addition, the role of calcium as a "third messenger" that provides a mechanism for interaction between phosphoinositide second messengers and components of the cyclic AMP signaling pathway is also discussed.

• Morris JC, Ping-Sheng L, Zhai HX, Shen TY, Mensa-Wilmot K
• Phosphatidylinositol phospholipase C is activated allosterically by the aminoglycoside G418. 2-deoxy-2-fluoro-scyllo-inositol-1-O-dodecylphosphonate and its analogs inhibit glycosylphosphatidylinositol phospholipase C.
• J Biol Chem. 1996; 271: 15468-77
• Display abstract

• Phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus cereus is inhibited by myo-inositol-1-O-dodecylphosphonate (Ins-1-O-dodecylphosphonate) (Morris, J. C., Ping-Sheng, L., Shen, T. Y., and Mensa-Wilmot, K.(1995) J. Biol. Chem. 270, 2517-2524). A set of novel fluorinated 2-deoxy-Ins-1-O-dodecylphosphonates were tested against PI-PLC, with potent competitive inhibition by 2-deoxy-2-fluoro-scyllo-Ins-1-O-dodecylphosphonate (VP-616L) (Xi(50) = 0.09). 2-Deoxy-2-fluoro-myo-Ins-1-O-dodecylphosphonate and 2-deoxy-2,2-difluoro-myo-Ins-1-O-dodecylphosphonate were 8.3-fold and 4.8-fold less effective, respectively, than VP-616L. Methyl 2-deoxy-2,2-difluoro-myo-Ins-1-O-dodecylphosphonate was inactive. Also, a hundredfold less PI-PLC is required to cleave a glycosylphosphatidylinositol (GPI) than is needed to cleave PI. Implied in these observations are the following: (i) in powerful inhibitors an active site residue probably interacts with the equatorially oriented fluoro substituent; (ii) substrate recognition requires a negative charge on the phosphoryl at the Ins-1 position, and (iii) a GPI is better substrate than PI, for PI-PLC. Aminoglycoside antibiotics kanamycin A, gentamycin, and G418 stimulated PI-PLC cleavage of the GPI anchor of variant surface glycoprotein (VSG) from Trypanosoma brucei 2- to 4-fold. G418, which appears to act on the enzyme.substrate complex, increased kcat and Km 6.4-fold and 9.9-fold, respectively. PI-PLC was activated by G418 even in the presence of the inhibitor VP-616L. In control experiments, the lectin concanavalin A (ConA), which probably acts by substrate sequestration, inhibited both PI-PLC (Xi(50) = 0.00025) and GPI-specific phospholipase D (Xi(50) = 0.00018). G418 failed to activate PI-PLC when ConA was present. These observations indicate that G418 is an allosteric activator of Bacillus cereus PI-PLC. Since G418 stimulates a purified enzyme that is not involved in aminoglycoside metabolism, we propose that binding of aminoglycosides to cellular proteins could contribute to the development of the nephrotoxicity associated with the use of these aminoglycoside antibiotics.

• Irvine R
• Phospholipid signalling. Taking stock of PI-PLC.
• Nature. 1996; 380: 581-3

• Kim CG, Park D, Rhee SG
• The role of carboxyl-terminal basic amino acids in Gqalpha-dependent activation, particulate association, and nuclear localization of phospholipase C-beta1.
• J Biol Chem. 1996; 271: 21187-92
• Display abstract

• The phospholipase C (PLC)-beta isozymes differ from the PLC-gamma and PLC-delta isozymes in that they possess a long COOH-terminal sequence downstream of their catalytic domain, are activated by alpha subunits of the Gq class of G proteins, associate with the particulate subcellular fraction, and are present in the nucleus. Most of the COOH-terminal domain of PLC-beta isozymes is predicted to be helical, and three regions in this domain, PLC-beta1 residues 911-928 (region 1), 1055-1072 (region 2), and 1109-1126 (region 3), contain a high proportion of basic residues that are highly conserved. Projection of the sequences of these three regions in helical wheels reveals clustering of the basic residues. The role of the COOH terminus and the clustered basic residues in PLC-beta1 was investigated by either truncating the entire COOH-terminal domain (mutant DeltaC) or replacing two or three clustered basic residues with isoleucine (or methionine), and expressing the mutant enzymes in CV-1, Rat-2, or Swiss 3T3 cells. The DeltaC mutant no longer showed the ability to be activated by Gqalpha, to translocate to the nucleus, or to associate with the particulate fraction. Substitution of clusters of basic residues in regions 1 and 2 generally reduced the extent of activation by Gqalpha, whereas substitution of a basic cluster in region 3 had no effect. Substitution of the cluster of lysine residues 914, 921, and 925 in region 1 had the most marked effect, reducing Gqalpha-dependent activity to 10% of that of wild type. All substitution mutants, with the exception of that in which lysine residues 1056, 1063, and 1070 in region 2 were substituted with isoleucine, behaved like the wild-type enzyme in showing an approximately equal distribution between cytoplasm and nucleus; only 12% of the region 2 mutant was present in the nucleus. None of the basic clusters appeared critical for particulate association; however, replacement of each cluster reduced the amount of PLC-beta1 in the particulate fraction by some extent, suggesting that all the basic residues contribute to the association, presumably by interacting with acidic residues in the particulate fraction. Membrane localization of PLC-beta isozymes is therefore likely mediated by both the COOH-terminal domain and the pleckstrin homology domain, the latter of which is known to bind phosphatidylinositol 4,5-biphosphate.

• Heinz DW, Ryan M, Smith MP, Weaver LH, Keana JF, Griffith OH
• Crystal structure of phosphatidylinositol-specific phospholipase C from Bacillus cereus in complex with glucosaminyl(alpha 1-->6)-D-myo-inositol, an essential fragment of GPI anchors.
• Biochemistry. 1996; 35: 9496-504
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• Numerous proteins on the external surface of the plasma membrane are anchored by glycosylated derivatives of phosphatidylinositol (GPI), rather than by hydrophobic amino acids embedded in the phospholipid bilayer. These GPI anchors are cleaved by phosphatidylinositol-specific phospholipases C (PI-PLCs) to release a water-soluble protein with an exposed glycosylinositol moiety and diacylglycerol, which remains in the membrane. We have previously determined the crystal structure of Bacillus cereus PI-PLC, the enzyme which is widely used to release GPI-anchored proteins from membranes, as free enzyme and also in complex with myo-inositol [Heinz, D.W., Ryan, M. Bullock, T.L., & Griffith, O. H. (1995) EMBO J. 14, 3855-3863]. Here we report the refined 2.2 A crystal structure of this enzyme complexed with a segment of the core of all GPI anchors, glucosaminyl(alpha 1-->6)-D-myo-inositol [GlcN-(alpha 1-->6)Ins ]. The myo-inositol moiety of GlcN(alpha 1-->6)Ins is well-defined and occupies essentially the same position in the active site as does free myo-inositol, which provides convincing evidence that the enzyme utilizes the same catalytic mechanism for cleavage of PI and GPI anchors. The myo-inositol moiety makes several specific hydrogen bonding interactions with active site residues. In contrast, the glucosamine moiety lies exposed to solvent at the entrance of the active site with minimal specific protein contacts. The glucosamine moiety is also less well-defined, suggesting enhanced conformational flexibility. On the basis of the positioning of GlcN(alpha 1-->6)Ins in the active site, it is predicted that the remainder of the GPI-glycan makes little or no specific interactions with B. cereus PI-PLC. This explains why B. cereus PI-PLC can cleave GPI anchors having variable glycan structures.

• Pawelczyk T
• [Phosphatidylinositol-specific phospholipase C isozymes and regulation of their activity]
• Postepy Biochem. 1996; 42: 290-8

• Horstman DA, DeStefano K, Carpenter G
• Enhanced phospholipase C-gamma1 activity produced by association of independently expressed X and Y domain polypeptides.
• Proc Natl Acad Sci U S A. 1996; 93: 7518-21
• Display abstract

• The X and Y domains of phospholipase C (PLC)-gamma1, which are conserved in all mammalian phosphoinositide-specific PLC isoforms and are proposed to interact to form the catalytic site, have been expressed as individual hexahistidine-tagged fusion proteins in the baculovirus system. Following coinfection of insect cells with recombinant viruses, association of X and Y polypeptides was demonstrated in coprecipitation assays. When enzyme activity was examined, neither domain possessed catalytic activity when expressed alone; however, coexpression of the X and Y polypeptides produced a functional enzyme. This reconstituted phospholipase activity remained completely dependent on the presence of free Ca2+. The specific activity of the X:Y complex was significantly greater (20- to 100-fold) than that of holoPLC-gamma1 and was only moderately influenced by varying the concentration of substrate. The enzyme activities of holoPLC-gamma1 and the X:Y complex exhibited distinct pH optima. For holoPLC-gamma1 maximal activity was detected at pH 5.0, while activity of the X:Y complex was maximal at pH 7.2.

• Milting H, Heilmeyer LM Jr, Thieleczek R
• Cloning of a phospholipase C-delta 1 of rabbit skeletal muscle.
• J Muscle Res Cell Motil. 1996; 17: 79-84
• Display abstract

• The phospholipase C isoform responsible for the increase in the total myoplasmic inositol 1,4,5-trisphosphate concentration during tetanic contraction of isolated skeletal muscle and its mechanism of activation is not known. We have cloned and sequenced a phospholipase C cDNA of rabbit skeletal muscle coding for a protein of 745 amino acids with a molecular mass of 84,440 kDa. The deduced amino acid sequence exhibits the phospholipase C-specific domains X and Y which according to current knowledge very likely represent the catalytic centre of the enzyme. An overall sequence homology of 88% to the phospholipase C-delta 1 of rat brain suggests that the encoded protein represents a phospholipase C-delta 1 isoform of rabbit skeletal muscle. Northern blot analysis shows, that this phospholipase C-delta is dominantly expressed in skeletal muscle, less strongly in smooth muscle (uterus) and lung and weakly in heart, kidney and brain. In the N-terminal part of the primary structure a consensus sequence for a canonical EF-hand Ca2+ binding domain can be identified together with a short positively charged motif which recently has been suggested to be essential for the binding of phosphatidylinositol 4,5-bisphosphate. If these two domains which are unique for phospholipase C-delta are sufficient in establishing a mechanism for the activation of the enzyme, inositol 1,4,5-trisphosphate formation in skeletal muscle could be the consequence of an increase in myoplasmic Ca2+.

• Exton JH
• Regulation of phosphoinositide phospholipases by hormones, neurotransmitters, and other agonists linked to G proteins.
• Annu Rev Pharmacol Toxicol. 1996; 36: 481-509
• Display abstract

• The actions of many hormones, neurotransmitters, and growth factors are mediated by the hydrolysis of phosphatidylinositol 4,5-bisphosphate catalyzed by specific isozymes of phospholipase C. This hydrolysis releases inositol 1,4,5-trisphosphate, which mobilizes Ca2+ ions from components of the endoplasmic reticulum, and 1,2-diacylglycerol, which activates isozymes of protein kinase C. The hormones and neurotransmitters activate beta-isozymes of phospholipase C through receptors that have seven transmembrane segments and couple to G proteins of the Gq and Gi/o families. Activation of phospholipase C by the Gq family involves their alpha-subunits, whereas activation by the Gi/o family involves their beta gamma-subunits. The growth factors activate gamma-isozymes of phospholipase C through receptors that become autophosphorylated due to their stimulated tyrosine kinase activity and provide binding sites for the Src homology domains of the isozymes. The molecular mechanisms by which agonists activate phopholipase isozymes are described in detail.

• Prufer K, Jirikowski GF
• Liposomal incorporation changes the effect of 1.25-dihydroxyvitamin D3 on the phospholipase C signal transduction pathway and the eicosanoid cascade on keratinocytes in vitro.
• Biochem Pharmacol. 1996; 51: 247-52
• Display abstract

• 1.25-dihydroxyvitamin D3 is of clinical importance (e.g. in the treatment of psoriasis) given its ability to regulate the proliferation and differentiation of human keratinocytes. 1.25-Dihydroxyvitamin D3 mediates its action via genomic and nongenomic pathways. The nongenomic actions begin with the activation of phospholipase C and the subsequent rapid rise in calcium within the cells. We incorporated 1.25-dihydroxyvitamin D3 in liposomes of varying compositions in an attempt to improve their effect/negative side effect ratio. The influence of empty liposomes (1 mM) and free and liposomally incorporated 1.25-dihydroxyvitamin D3 (10 nM) on the rapid release of sulfidoleucotrien and inositole 1,4,5 triphosphate was examined in keratinocytes in vitro. Free 10 nM 1.25-dihydroxyvitamin D3 provoked a rapid rise in sulfidoleucotriens within 30 seconds, followed by a swift decrease in sulfidoleucotrien and inositole 1,4,5-triphosphate concentration after 10 minutes. Empty liposomes and liposomal-incorporated 1.25-dihydroxyvitamin D3 did not show such a strong effect. These results suggest the occurrence of specific binding sites for 1.25-dihydroxyvitamin D3 on the membrane level that are incapable of recognizing 1.25-dihydroxyvitamin D3 trapped within liposomal membrane.

• LaBelle EF, Polyak F
• Phospholipase C beta 2 in vascular smooth muscle.
• J Cell Physiol. 1996; 169: 358-63
• Display abstract

• Receptor-mediated inositol 1,4,5-trisphosphate formation in most tissue is dependent on a variety of phospholipase C isoforms. To determine which phospholipase C isoforms were present in vascular smooth muscle compared to brain, liver, and spleen, we extracted proteins from these tissues and separated and identified the phospholipase C isoforms by immunoblotting. Aliquots of rat tail artery were examined by this procedure, together with aliquots of rat liver, spleen, cerebral cortex, hippocampus, cerebellum, aorta, and mesenteric artery. Phospholipase C gamma 1 was shown to be present in all of these tissues, while phospholipase C beta 1 was shown to be limited to fractions from brain. Phospholipase C delta 1 was detected in rat tail artery, mesenteric artery, aorta, and brain. Phospholipase C beta 2 was found in rat tail artery, liver, and brain. This is the first report of phospholipase C beta 2 in tissues other than HL60 cells. Since G proteins activate IP3 production via stimulation of phospholipase C beta isoforms in many tissues, and agonist-stimulated IP3 production in smooth muscle requires G protein activation, phospholipase C beta 2 may be required for agonist-stimulated force production in vascular smooth muscle.

• Amtmann E
• The antiviral, antitumoural xanthate D609 is a competitive inhibitor of phosphatidylcholine-specific phospholipase C.
• Drugs Exp Clin Res. 1996; 22: 287-94
• Display abstract

• The effect of the antiviral, antitumoural xanthate D609 on the activity of phospholipase A2, C (PC- and Pi-specific) and D was investigated. D609 is the first model substance of a new concept of antiviral therapy that interferes with cellular regulation mechanisms, rather than with virus coded enzymes. Exclusively phosphatidylcholine (PC) specific phospholipase C (PC-PLC) was found to be inhibited in a dose-dependent manner. Enzyme activity was determined either as the rate of acid release from PC or as the rate of phosphorylcholine production form 3H labelled PC. Lineweaver-Burk plots revealed D609 as a competitive inhibitor of PC-PLC with a Ki of 6.4 microM. In addition, D609 competitively inhibited PC-PLC mediated cleavage of P-nitrophenylphosphorylcholine (p-NPP), a pseudo-substrate of PC-PLC with a Ki of 8.8 microM. These data suggest that D609 competes with the phosphorylcholine residue of PC for binding to PC-PLC.

• Beaven MA
• Calcium signalling: sphingosine kinase versus phospholipase C?
• Curr Biol. 1996; 6: 798-801
• Display abstract

• A recent study shows that sphingosine kinase and its lipid product have an essential signalling function; they act in the mobilization of calcium ions in antigen-stimulated mast cells. This finding may have relevance to signalling in other cells of the immune system.

• Leonis MA, Silbert DF
• Genomic organization of the hamster phospholipase C-delta 1 gene: differential loss of separate alleles of the phospholipase C-delta 1 gene in two fibroblast mutants lacking phospholipase C-delta 1.
• Biochem Biophys Res Commun. 1996; 224: 382-90
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• In earlier work from this laboratory, mutant Chinese hamster lung fibroblasts D1-6b and D1-9b were shown to lack phosphatidylinositol-specific phospholipase C-delta 1 (PLC delta 1). In the current study, Northern analysis of mRNA samples from these mutants fails to detect full-length transcripts for PLC delta 1. Southern analysis of Sac1 digests of wild type and mutant cell genomic DNA hybridized against probe made to full-length rat brain PLC delta 1 cDNA suggested that these mutants differ from one another and wild type controls. This observation is shown to be the result of the differential loss, in both mutants, of one of two alleles for the hamster PLC delta 1 gene normally present in the parental cell line. During the course of these studies, the hamster PLC delta 1 gene was cloned and sequenced. This genomic sequence spans 13 kb of DNA and encoded 98.5% of the predicted coding sequence for the hamster PLC delta 1 cDNA within 15+ exons.

• Paulssen RH, Woodson J, Liu Z, Ross EM
• Carboxyl-terminal fragments of phospholipase C-beta1 with intrinsic Gq GTPase-activating protein (GAP) activity.
• J Biol Chem. 1996; 271: 26622-9
• Display abstract

• Fragments of the approximately 50 kDa COOH-terminal region of phospholipase C-beta1 (PLC-beta1(1)), ranging in size from 14 to 38 kDa, were expressed in Escherichia coli, purified, and tested for their regulatory activities. As expected, none of the fragments had phospholipase activity. Several fragments, referred to as PLC tails, displayed GTPase-activating protein (GAP) activity for Gq, the G protein class that stimulates the PLC-betas in response to receptors. Gq GAP activity is characteristic of intact PLC-betas. In reconstituted phospholipid vesicles that contained purified Gq and m1 muscarinic cholinergic receptors, the most active tails increased agonist-stimulated, steady-state GTPase activity over 4-fold. Stimulation of steady-state GTPase by the tails depended on receptors for facilitation of GDP-GTP exchange, suggesting that the tails act by accelerating hydrolysis of bound GTP. In addition to intrinsic GAP activity, one tail with high GAP activity and others with low or minimal activity potentiated the GAP activity of intact PLC-beta1. Other tails inhibited PLC-beta1s GAP effect. Both intrinsic GAP activity and potentiation of the PLC-beta1 GAP effect were often biphasic, with maxima as low as 100 nM tail and declining activities at higher concentrations. Several tails inhibited either the phospholipase activity of PLC-beta1, its stimulation by Gq, or both. The tails thus define the region of PLC-beta1 that has Gq GAP activity and suggest a mechanism of action in which the COOH terminus of PLC-betas can interact with Gq and with other PLC-beta1 molecules.

• Basanez G, Nieva JL, Goni FM, Alonso A
• Origin of the lag period in the phospholipase C cleavage of phospholipids in membranes. Concomitant vesicle aggregation and enzyme activation.
• Biochemistry. 1996; 35: 15183-7
• Display abstract

• When phospholipase C is added to a suspension of large unilamellar vesicles of egg phosphatidylcholine, maximal rates of hydrolysis occur only after a latency period. No lag period is seen when the substrate is in the form of small (sonicated) vesicles, or of short-chain phosphatidylcholine monomers. For a given vesicle concentration, the lag time may vary as a function of Ca2+, enzyme concentration, or temperature, but activation occurs at a fixed molar fraction of diacylglycerol produced. Lag times decrease gradually with vesicle size, and also with the amount of diacylglycerol present in the bilayers when it is mixed with phospholipid prior to enzyme addition. Parallel recordings of enzyme activity and suspension turbidity reveal that in all cases the latency period ends concomitantly with the start of a process of vesicle aggregation. Both the lag time and the amount of diacylglycerol formed before activation decrease with vesicle concentration, suggesting that enzyme activation is somehow related to vesicle aggregation. The latency period of phospholipase C may be explained in terms of a hypothesis according to which (a) full enzyme activity requires the presence of membrane surface irregularities or defects, (b) the diacylglycerol generated in the lag phase produces some kind of phase separation, with the formation of diacylglycerol-rich "patches" or domains, (c) vesicles aggregate through contacts between those patches, and (d) aggregation causes (and/or increases, and/or stabilizes) the surface inhomogeneities that allow fast enzyme activity. These data and suggestions may be relevant to the process of model membrane fusion promoted by phospholipase C.

• Kobayashi T, Kishimoto M, Okuyama H
• Phospholipases involved in lysophosphatidylinositol metabolism in rat brain.
• J Lipid Mediat Cell Signal. 1996; 14: 33-7
• Display abstract

• A phospholipase C activity highly specific for lysophosphoinositide (lysoPI-PLC) has been demonstrated to be present in synaptic plasma membranes of the rat brain. Several lines of evidence suggested that the lysoPI-PLC is an enzyme distinct from known isoforms of phosphoinositide-specific phospholipase C (PI-PLC). On the other hand, the occurrence of a Ca(2+)-independent phospholipase A1 hydrolyzing PI in rat brain was also demonstrated. The lysoPI-PLC hydrolyzed the 2-acyl isomer as well as the 1-acyl isomer of lysoPI. These findings suggest possible pathways for PI metabolism through lysoPI to yield monoacylglycerol (mainly 2-arachidonoyl glycerol) and inositolphosphates in the brain, which are different from the well-characterized PI-PLC pathway.

• Revin VV, Nabokina SM, Anisimova IA, Gruniushkin IP
• [Activity of phosphoinositide-specific phospholipase C in rabbit nerves at rest and during stimulation]
• Biokhimiia. 1996; 61: 815-20
• Display abstract

• The dependence of rabbit sciatic nerve PtdIns-specific phospholipase C activity Ca2+ and pH has been studied. The enzyme was found to hydrolyze PtdInsP2 in a Ca(2+)-dependent manner. The maximal activity was detected at 1mM CaCl2 for the cytosolic enzyme and at 0.1 mM CaCl2 for membrane-bound enzyme. The pH optima determined for the cytosolic and particulate enzymes were pH 6.0 and 7.0, respectively. The enzyme activity in the cytosolic fraction was shown to remain practically constant under nerve stimulation (200 impulses/sec, 5 min), whereas in microsomal fraction it was 44% greater compared to the resting nerve. It is suggested that the enzyme is bound to membrane in intact axon, therefore the rapid signal (depolarizing stimulus) transduction into axon becomes possible.

• Grobler JA, Essen LO, Williams RL, Hurley JH
• C2 domain conformational changes in phospholipase C-delta 1.
• Nat Struct Biol. 1996; 3: 788-95
• Display abstract

• The structure of the PH-domain truncated core of rat phosphoinositide-specific phospholipase C-delta 1 has been determined at 2.4 A resolution and compared to the structure previously determined in a different crystal form. The stereochemical relationship between the EF, catalytic, and C2 domains is essentially identical. The Ca2+ analogue Sm3+ binds at two sites between the jaws of the C2 domain. Sm3+ binding ejects three lysine residues which bridge the gap between the jaws and occupy the Ca2+ site in the apoenzyme, triggering a conformational change in the jaws. The distal sections of the C2 jaws move apart, opening the mouth by 9 A and creating a gap large enough to bind a phospholipid headgroup.

• Ryan M, Smith MP, Vinod TK, Lau WL, Keana JF, Griffith OH
• Synthesis, structure-activity relationships, and the effect of polyethylene glycol on inhibitors of phosphatidylinositol-specific phospholipase C from Bacillus cereus.
• J Med Chem. 1996; 39: 4366-76
• Display abstract

• Substrate analog inhibitors of Bacillus cereus phosphatidylinositol-specific phospholipase C (PI-PLC) were synthesized and screened for their suitability to map the active site region of the enzyme by protein crystallography. Analogs of the natural substrate phosphatidylinositol (PI) were designed to examine the importance of the lipid portion and the inositol phosphate head group for binding to the enzyme. The synthetic compounds contained pentyl, hexyl, or hexanoyl and octyl lipid chains at the sn-1 and sn-2 positions of the glycerol backbone and phosphonoinositol, phosphonic acid, methyl phosphonate, phosphatidic acid, or methyl phosphate at the sn-3 position. The most hydrophobic compound, dioctyl methyl phosphate 14, was also the best inhibitor with an IC50 of 12 microM. In a series of dihexyl lipids, compounds with phosphonoinositol head groups inhibited more strongly than those that do not contain inositol but are otherwise identical. Compound 29, a short-chain lipid with a phosphonoinositol head group, was found to be a competitive inhibitor and the most potent in this series with an IC50 of 18 microM (Ki = 14 microM). Analogs with dihexyl chains were better inhibitors than those with dihexanoyl chains, presumably because the ether-linked lipids are more hydrophobic than the ester-linked lipids. No appreciable difference in inhibition was found between a phosphonoinositol lipid and the corresponding difluorophosphonoinositol lipid. Inositols and inositol derivatives that do not contain lipid moieties show IC50s about 3 orders of magnitude above those of the short-chain lipids. In this group, glucosaminyl(alpha 1-->6)-D-myo-inositol inhibited more strongly than myo-inositol, which in turn is a better inhibitor than inositol phosphate. The addition of polyethylene glycol (PEG-600) resulted in a marked decrease in inhibition by the short-chain lipids, but had little effect on the water-soluble head group analogs. This is accounted for in terms of solubilization of the amphipathic inhibitors by PEG. Since PEG is required in the crystallization, these data indicate that the best strategy for obtaining enzyme inhibitor complexes is to start by cocrystallizing PI-PLC with the head group analogs. The next step is to synthetically add the shortest possible hydrophobic moieties to the analogs and cocrystallize these with the enzyme. This strategy may be applicable to other lipolytic enzymes.

• van Heugten HA, Eskildsen-Helmond YE, de Jonge HW, Bezstarosti K, Lamers JM
• Phosphoinositide-generated messengers in cardiac signal transduction.
• Mol Cell Biochem. 1996; 157: 5-14
• Display abstract

• A multitude of agonists like e.g. endothelin-1, angiotensin-II, serotonin, thrombin, histamine and vasopressin as well as alpha 1-adrenergic and muscarinic stimulation lead to stimulation of the phosphoinositide cycle in the heart. Besides the seven membrane spanning-domain receptor-coupled stimulation of the key enzyme of the phosphoinositide cycle, phospholipase C-beta, another class of hormones, growth factors, also couple to the phosphoinositide cycle, now through receptors with intrinsic tyrosine kinase activity that can phosphorylate and stimulate the phospholipase C-gamma isozyme. In this review we summarize the multitude of receptor (sub)types, G-protein-subunit- and phospholipase C-isozymes that are present in the heart. Furthermore, generation of second messengers and cellular responses are described together with the (patho)physiological implications for the heart of phosphoinositide cycle activation and second messenger accumulation.

• Wang LP, Lim C, Kuan Y, Chen CL, Chen HF, King K
• Positive charge at position 549 is essential for phosphatidylinositol 4,5-bisphosphate-hydrolyzing but not phosphatidylinositol-hydrolyzing activities of human phospholipase C delta1.
• J Biol Chem. 1996; 271: 24505-16
• Display abstract

• Point mutagenesis, phosphatidylinositol (PI), and phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis assays and equilibrium centrifugation PIP2 assays were used to study the functional roles of four highly conserved arginine residues in the Y region of human phospholipase C delta1 (PLCdelta1) (Arg-527, -549, -556, -701). Most of the mutant enzymes were either partially defective or fully active in their abilities to catalyze the hydrolysis of PI or PIP2. However, upon substitution of Arg-549 by glycine or histidine, the mutant enzyme was defective in its ability to catalyze the hydrolysis of PIP2, but it is still able to hydrolyze PI. Replacing Arg-549 with lysine had little effect on the level of PI and PIP2 hydrolytic activities of the mutant enzyme. The residual PIP2 hydrolyzing activity of R549H is highly dependent on pH. R549H showed 5-10% of the PIP2-hydrolyzing activity of the native enzyme between pH 5 and 7 and nondetectable PIP2-hydrolyzing activity at pH 8. The PIP2-hydrolyzing activity of R549G was not detectable at all pH values. Kinetic analysis of PLCdelta1-catalyzed PIP2 hydrolysis revealed that the micellar dissociation constant Ks and interfacial Michaelis constant Km were similar in the native, R549K, and R549H enzymes; but the specific activity at the saturated substrate mole fraction and infinite level of substrate (Vmax) of the R549H mutant were reduced by a factor of 15. PIP2 competitively inhibits the native enzyme to hydrolyze PI at both pH 7 and 8. However, PIP2 inhibits R549H only at pH 7.0 and does not inhibit R549G at either pH. Taken together, these results suggest that positive charge at position 549 of PLCdelta1 protein is essential for the enzyme to recognize and catalyze the hydrolysis of PIP2 but not PI.

• Hwang SC, Park KH, Ha MJ, Noh IS, Park TB, Lee YH
• Distribution of phospholipase C isozymes in normal human lung tissue and their immunohistochemical localization.
• J Korean Med Sci. 1996; 11: 305-13
• Display abstract

• Phospholipase C(PLC) plays a central role in signal transduction and it is important in cellular growth, differentiation and transformation. There are currently ten known mammalian isozymes of PLC identified and cloned. However, there are no report of PLC distribution in human lung tissue or their significances in pulmonary diseases. Presence of various PLC isozymes in normal human lung tissue was studied from surgical specimens. PLC isozymes in tissue extracts of the lung were partially purified by successive chromatographic steps on heparin-sepharose CL-6B conventional and TSKgel heparin-5PW HPLC columns and their activities were assayed. PLC activity peaks identified in the chromatography were immunoblotted with specific antibodies against ten known mammalian PLC isozymes(PLC-beta 1-4, -gamma 1-2, and -delta 1-4). In addition, immunohistochemical staining of the lung tissue was performed to determine subcellular and histological localization of PLC isozymes. The results indicate that normal human lungs contain beta 1, beta 3, gamma 1, and delta 1, isozymes of PLC. The order of amount present in the lung tissue was PLC-delta 1 > gamma 1 > beta 1 >> beta 3, in descending order. On immunohistochemistry, PLC-gamma 1 was most widely distributed and was present in bronchiolar epithelium, in type I and type II pneumocytes as well as in fibroblasts of the interstitial tissue. PLC-delta 1 was present in the cytoplasm of the bronchiolar epithelium whereas PLC-beta 1 was localized to the apical membranous portion of the same epithelium. PLC-beta 3 was seen in the nucleus of the respiratory and alveolar lining epithelium as well as in the nucleus of lung fibroblasts.

• Moser C, Roth HP, Kirchgessner M
• Influence of alimentary zinc deficiency on the concentration of the second messengers D-myo-inositol-1,4,5-trisphosphate (IP3) and s,n-1,2-diacylglycerol (DAG) in testes and brain of force-fed rats.
• Biol Trace Elem Res. 1996; 52: 281-91
• Display abstract

• The phosphatidylinositol-specific phospholipase C, presumably a Zn-metalloenzyme, catalyzes the hydrolysis of phosphatidylinositol-4,5-bisphosphate to inositol-1,4,5-trisphosphate (IP3) and s,n-1,2-diacylglycerol (DAG). The activity of phosphatidylinositol-specific phospholipase C was measured indirectly by determination of the metabolites IP3 and DAG in Zn deficiency. For this purpose 24 male Sprague-Dawley rats with an average live mass of 117 g were divided into 2 groups of 12 animals each. The Zn-deficient and the control group received a semisynthetic casein diet with a Zn content of 1.6 ppm and 115 ppm, respectively. In order to prevent the reduced feed intake that occurs in Zn deficiency and the associated energy and protein depletion from interfering with the experimental parameters, all animals were fed four times daily by gastric tube. This made it possible to supply all animals with adequate nutrients and to synchronize the feed intake exactly. After 12 d, the depleted rats were in a severe state of Zn deficiency, as demonstrated by the reduction of Zn in the serum and the femur by 74% and 43%, respectively, and the 28% lower serum activity of alkaline phosphatase. The radioimmunologically determined concentrations of IP3 were reduced by a significant 53% in the testes of the Zn-deficient rats (0.24 nmol IP3/g wet wt) compared to the control animals (0.51 nmol IP3/g wet wt), while the IP3 concentration in the brain was not affected by the alimentary Zn supply (1.7 and 1.6 nmol IP3/g wet wt, respectively). The DAG concentrations in the testes (474 vs 471 nmol DAG/g wet wt) and the brain (594 vs 640 nmol DAG/g wet wt), which were determined by radioenzymatic methods, showed no significant differences in relation to the alimentary Zn supply. The fact that the Zn concentration in the Zn-deficient rats was reduced only in the testes and not in the brain and that high concentrations of DAG may also result from other metabolic processes suggests that the phosphatidylinositol-specific phospholipase C in the mammalian organism is a Zn-metalloenzyme whose activity is reduced in alimentary Zn deficiency in tissues suffering Zn loss.

• Kobayashi T, Tamura H, Taguchi R, Udaka S, Ikezawa H
• High-level expression of Bacillus thuringiensis phosphatidylinositol-specific phospholipase C by the Bacillus brevis host-vector system.
• Jpn J Med Sci Biol. 1996; 49: 103-12
• Display abstract

• We succeeded in hyperproduction of Bacillus thuringiensis phosphatidylinositol-specific phospholipase C (PIPLC), using a Bacillus brevis 47 expression system. The recombinant B. thuringiensis PIPLC was expressed under the control of the middle wall protein gene promoter in B. brevis expression vector pNU211. A large amount of recombinant PIPLC (0.4 g per liter culture) was secreted into the medium as a mature enzyme, and the enzymatic properties of purified recombinant PIPLC were similar to those of the enzyme from wild-type B. thuringiensis. This system provides a useful approach to the three-dimensional structure-function relationship of PIPLC.

• Scarlata S et al.
• Inhibition of phospholipase C-delta 1 catalytic activity by sphingomyelin.
• Biochemistry. 1996; 35: 14882-8
• Display abstract

• We measured the ability of sphingomyelin (SPM) to inhibit phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] hydrolysis catalyzed by human phospholipase C-delta 1 (PLC-delta 1) in model membranes and detergent phospholipid mixed micelles. SPM strongly inhibited PLC-delta 1 catalytic activity measured in large unilamellar vesicles (LUVs) composed of egg phosphatidylcholine (PC), PI(4,5)P2, and SPM from brain or egg. At 37 or 45 degrees C, the rate of PI(4,5)P2 hydrolysis in PC/SPM/PI(4,5)P2 vesicles (15:80:5 mol:mol) was less than 25% of that observed in PC/PI(4,5)P2 vesicles (95:5). By contrast, catalysis was only weakly inhibited by equivalent concentrations of the SPM analog, 3-deoxy-2-O-stearoyl-SPM, which lacks hydrogen bond-donating groups at the C-3 and C-2 positions of the sphingolipid backbone. Inhibition by SPM was not observed in detergent/phospholipid mixed micelles. The binding affinity of PLC-delta 1 for vesicles containing PC and PI(4,5)P2 was slightly diminished by inclusion of SPM in the lipid mixture, but not enough to account for the decreased rate of catalysis. We could find no evidence of specific binding of the enzyme to SPM, which argues against a simple negative allosteric mechanism. To understand the cause of inhibition, the effects of SPM and 3-deoxy-2-O-stearoyl-SPM on the bulk properties of the substrate bilayers were examined. Increasing the mole fraction of SPM altered the fluorescence emission spectra of two sets of head group probes, 6-lauronyl(N,N-dimethylamino)naphthalene and N-[5-(dimethylamino)naphthalene-1-sulfonyl]-1,2-dihexadecanoyl-sn- glycero-3-phosphoethanolamine, that are sensitive to water content at the membrane/solution interface. Results obtained with both probes suggested a reduction in hydration with increasing SPM content. Vesicles containing 3-deoxy-2-O-stearoyl-SPM produced intermediate changes. Our results are most consistent with a model in which SPM inhibits PLC by increasing interlipid hydrogen bonding and by decreasing membrane hydration; both factors raise the energy barrier for activation of PLC-delta 1 at the membrane/protein microinterface.

• Roberts MF, Wu Y, Zhou C, Geng D, Tan C
• Mechanism and structure based inhibitors of phospholipase C enzymes.
• Adv Enzyme Regul. 1996; 36: 57-71
• Display abstract

• PI-specific PLC enzymes are a key component of phosphatidylinositol-mediated signaling pathways since the hydrophobic product, diacylglycerol, activates protein kinase C and the water-soluble product, inositol trisphosphate, is involved in Ca2+ mobilization. Nonspecific, or PC-PLC, enzymes can generate diacylglycerol without Ca2+ mobilization. A series of inhibitors, both lipophilic and water-soluble, have been synthesized to target each of these two classes of PLC enzymes. Design of the inhibitors was based on proposed enzyme mechanisms and available crystal structures. The solution conformations of the lipophilic phospholipid analogs, (diheptanoylphosphatidyl(2-O-methyl)inositol for PI-PLC and a dihexanoyl-sn-(3-N-benzylaminoglycero)phosphoramidocholine for PC-PLC, have been determined using NMR methodology and the interaction of these compounds with bacterial enzymes has been examined. Water-soluble inhibitors include strained cyclic phosphonates for PI-PLC and vanadate for PC-PLC. An eventual goal of this work is to generate compounds that specifically target each type of intracellular PLC activity.

• Daniele JJ, Maggio B, Bianco ID, Goni FM, Alonso A, Fidelio GD
• Inhibition by gangliosides of Bacillus cereus phospholipase C activity against monolayers, micelles and bilayer vesicles.
• Eur J Biochem. 1996; 239: 105-10
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• The effect of complex glycosphingolipids (gangliosides) on the activity of phospholipase C from Bacillus cereus was studied using lipid monolayers, mixed micelles and small unilamellar vesicles containing phosphatidylcholine as substrate. In all artificial membrane systems assayed, gangliosides exhibit qualitatively similar inhibitory properties. Gangliosides decrease the enzyme activity irrespective of the aggregation structure in which the substrate is offered to B. cereus phospholipase C, and they do not affect the adsorption process of the enzyme. The modulatory effect of gangliosides occurs at the level of the interface, affecting both the maximum rate of catalysis of the enzyme already adsorbed and the availability of the substrate in a suitable organization for enzyme catalysis to take place.

• Roberts MF
• Phospholipases: structural and functional motifs for working at an interface.
• FASEB J. 1996; 10: 1159-72
• Display abstract

• Phospholipases form a ubiquitous class of enzymes optimized to catalyze the hydrolysis of phospholipids. Because their products are often second messengers, they are highly regulated by the cell. For a given ester bond, there are separate secreted as well as cytoplasmic phospholipases with different substrate specificities and modes of regulation. As it becomes available, structural information provides a view of interfacial catalysis for several of these phospholipases on a molecular level. Recent structural advances include solution structures of a pancreatic phospholipase A2 in the absence and presence of a micellar interface, crystal structures of a bacterial phosphatidylinositol-phospholipase C whose active site is reminiscent of ribonuclease, and a Ca2+ lipid binding domain with high homology to regions in several cytoplasmic phospholipases that can model the way those proteins interact with the membrane surface. Phospholipases also have a wide and complex array of regulatory mechanisms involving cytoplasmic proteins, notably G-proteins, as well as different effector lipids (e.g., phosphatidylinositol-4,5-biphosphate, or PIP2) or Ca2+. Deconvolution of these interactions is necessary to understand their roles in different signal transduction pathways.-Roberts, M. F. Phospholipases: structural and functional motifs for working at an interface.

• Bhatia J, Davies A, Gaudoin JB, Saibil HR
• Rhodopsin, Gq and phospholipase C activation in cephalopod photoreceptors.
• J Photochem Photobiol B. 1996; 35: 19-23
• Display abstract

• We present characterization of the rhodopsin, Gq and phosphatidylinositol-specific phospholipase C (PLC) from the signal transduction pathway of cephalopod photoreceptors. Cephalopod rhodopsins are unique in possessing a C-terminal extension of proline-rich repeats, and they have a strong tendency to form ordered arrays. Two-dimensional arrays of a full-length and C-terminally-truncated cephalopod rhodopsin have been obtained. The C termini appear to cluster the rhodopsins into small groups. An AlF4(-)-activated Gq alpha subunit has been isolated and shown to activate a partially purified PLC beta. This 130 kDa PLC, isolated by absorption on heparin agarose, showed a specific activity of 195 nmol of phosphatidylinositol 4,5-bisphosphate hydrolysed per milligram of protein per minute in the presence of 1.6 microM free calcium.

• Fankhauser H, Schweingruber AM, Edenharter E, Schweingruber ME
• Growth of a mutant defective in a putative phosphoinositide-specific phospholipase C of Schizosaccharomyces pombe is restored by low concentrations of phosphate and inositol.
• Curr Genet. 1995; 28: 199-203
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• A mutant (plc1-1) of Schizosaccharomyces pombe unable to grow on a minimal medium containing high amounts of phosphate was selected. On yeast-extract agar its growth is temperature sensitive. Tests in liquid synthetic medium show that growth of the mutant is partially restored by lowering the phosphate and inositol concentrations in the growth medium. The growth defect is fully suppressed by a plasmid encoding a putative protein having the structural features of phosphoinositide-specific phospholipases C (PI-PLC). This protein, of 899 amino-acids, contains the characteristic X and Y domains found in all PI-PLCs of higher and lower eucaryotes and reveals, in addition, an EF-hand motif (putative Ca(2+)-binding site). Like the corresponding enzyme from Saccharomyces cerevisiae, the S. pombe PI-PLC is most similar to the delta form of PI-PLC isoenzymes. The cloned gene integrates at the plc1 site indicating that plc1 codes for a putative PI-PLC. Plc1 physically maps on the left arm of chromosome II between rad11 and mei3.

• Mitchell R, Wolbers WB, Sim P, Fennell M
• The regulation of phospholipase C (PLC) and phospholipase D (PLD) by G protein receptor-activated tyrosine kinases in alpha T3-1 cells.
• Biochem Soc Trans. 1995; 23: 208-208

• Tsutsumi T, Kobayashi T, Miyashita M, Watanabe S, Homma Y, Okuyama H
• A lysophosphoinositide-specific phospholipase C distinct from other phospholipase C families in rat brain.
• Arch Biochem Biophys. 1995; 317: 331-6
• Display abstract

• Distinct phospholipase C activities capable of hydrolyzing lysophosphatidylinositol (lysoPI-PLC) or phosphatidylinositol (PI-PLC) have been demonstrated in rat brain membranes. Treatment of brain membranes with 1 M NaCl or 1% sodium cholate solubilized a majority of PI-PLC activity from the membranes, whereas a significant level of lysoPI-PLC activity still remained membrane-associated. Most of the lysoPI-PLC activity was recovered in a 0.5% sodium deoxycholate-0.25 M NaCl extract which contained only low levels of PI-PLC activity. Using the separated fractions, differences between lysoPI-PLC and the known PI-PLC isoforms were examined. A specific peptide inhibitor of PI-PLC, which was previously shown to interact with active site regions common to known PI-PLC activity. Immunoblot analysis of both the lysoPI-PLC-rich and PI-PLC-rich fractions revealed that an antiserum against PI-PLC delta 1 cross-reacted with other PI-PLC isoforms, but not significantly with lysoPI-PLC. Furthermore, lysoPI-PLC was more resistant to sulfhydryl reagents than was PI-PLC. Our results indicate that lysoPI-PLC is an enzyme distinct from PI-PLC and that lysoPI-PLC possesses a different active site than known PI-PLC isoforms.

• Lee SB, Rhee SG
• Significance of PIP2 hydrolysis and regulation of phospholipase C isozymes.
• Curr Opin Cell Biol. 1995; 7: 183-9
• Display abstract

• Phosphatidylinositol 4,5-bisphosphate (PIP2) is an important component of several intracellular signaling pathways. It serves as a substrate for phospholipase C, which produces the second messengers inositol 1,4,5-trisphosphate and diacylglycerol. It is also a substrate for a phosphatidylinositol 3-kinase, and regulates the function of a number of actin-binding proteins. PIP2 has been shown recently to serve as a cofactor for a phosphatidylcholine-specific phospholipase D and as a membrane-attachment site for many signaling proteins containing pleckstrin homology domains. The need to stringently regulate the cellular concentration of PIP2 is reflected in part by the fact that there are at least ten distinct mammalian phospholipase C isozymes and multiple mechanisms linking these isozymes to various receptors.

• Pizauro JM, Ciancaglini P, Leone FA
• Characterization of the phosphatidylinositol-specific phospholipase C-released form of rat osseous plate alkaline phosphatase and its possible significance on endochondral ossification.
• Mol Cell Biochem. 1995; 152: 121-9
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• Alkaline phosphatase activity was released up to 100% from the membrane by incubating the rat osseous plate membrane-bound enzyme with phosphatidylinositol-specific phospholipase C. The molecular weight of the released enzyme was 145,000 on Sephacryl S-300 gel filtration and 66,000 on PAGE-SDS, suggesting a dimeric structure. Solubilization of the membrane-bound enzyme with phospholipase C did not destroy its ability to hydrolyse PNPP, ATP and pyrophosphate. The hydrolysis of ATP and PNPP by phosphatidylinositol-specific phospholipase C-released enzyme exhibited 'Michaelian' kinetics with K0.5 = 70 and 979 microM, respectively. For pyrophosphate, K0.5 was 128 microM and site-site interactions were observed (n = 1.4). Magnesium ions were stimulatory (K0.5 = 1.5 mM) and zinc ions were a powerful noncompetitive inhibitor (Ki = 6.2 microM) of phosphatidylinositol-specific phospholipase C-released enzyme. Phosphatidylinositol-specific phospholipase C-released alkaline phosphatase was relatively stable at 40 degrees C. However, with increasing temperature from 40-60 degrees C, the enzyme was inactivated rapidly following first order kinetics and thermal inactivation constants varied from 5.08 x 10(-4) min-1 to 0.684 min-1. Treatment of phosphatydilinositol-specific phospholipase C-released alkaline phosphatase with Chellex 100 depleted to 5% its original PNPPase activity. Magnesium (K0.5 = 29.5 microM), manganese (K0.5 = 5 microM) and cobalt ions (K0.5 = 10.1 microM) restored the activity of Chelex-treated enzyme, demonstrating its metalloenzyme nature. The stimulation of Chelex-treated enzyme by calcium ions (K0.5 = 653 microM) was less effective (only 26%) and occurred with site-site interactions (n = 0.7). Zinc ions had no stimulatory effects. The possibility that the soluble form of the enzyme, detected during endochondral ossification, would arise by the hydrolysis of the Pl-anchored form of osseous plate alkaline phosphatase is discussed.

• Pillai S, Bikle DD, Su MJ, Ratnam A, Abe J
• 1,25-Dihydroxyvitamin D3 upregulates the phosphatidylinositol signaling pathway in human keratinocytes by increasing phospholipase C levels.
• J Clin Invest. 1995; 96: 602-9
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• 1,25-Dihydroxyvitamin D3 (1,25(OH)2D3) induces the differentiation of normal human keratinocytes, in part by increasing their basal intracellular calcium levels (Cai) over a period of hours. Agonists such as ATP acting through membrane receptors cause an immediate but transient increase in Cai accompanied by an increase in inositol trisphosphate (IP3). Treatment of keratinocytes for 24 h with 1 nM 1,25(OH)2D3 resulted in a two- to four-fold potentiation of the Cai response of these cells to ATP. This potentiation was inhibitable with cycloheximide, unaccompanied by a change in total intracellular calcium pools, but associated with an increase in basal IP3 levels and ATP-stimulated IP3 production. Treatment with 1,25(OH)2D3 raised the protein and mRNA levels of phospholipase C isoenzymes, particularly phospholipase C-beta 1 in a dose-dependent manner. These studies indicate that 1,25(OH)2D3 modulates the keratinocyte signal transduction pathway by induction of phospholipase isoenzymes, a previously undescribed action for this hormone.

• Manzoli L et al.
• Phosphoinositide signaling in nuclei of Friend cells: tiazofurin down-regulates phospholipase C beta 1.
• Cancer Res. 1995; 55: 2978-80
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• Previous investigations have demonstrated the presence of conventional lipid kinases and phospholipase C (PLC) activities in nuclei of Friend erythroleukemia cells. Moreover, when Friend erythroleukemia cells are treated for 96 h with the antitumor drug tiazofurin, the induction of erythroid differentiation is accompanied by changes in amounts of both phosphatidylinositol and phosphatidylinositol 4,5-bisphosphate due to the inhibition of an uncharacterized nuclear PLC activity. Here, we show that the nuclear PLC beta 1 isoform is down-regulated by tiazofurin (5 microM) treatment of Friend erythroleukemia cells as shown by both Western blot and Northern blot analyses for PLC beta 1 message. This indicates that PLC beta 1 down-regulation is tightly linked with erythroid differentiation of Friend erythroleukemia cells and that the autonomous nuclear signaling via inositol lipid cycle can be controlled by the antitumor drug tiazofurin.

• Hergenrother PJ, Spaller MR, Haas MK, Martin SF
• Chromogenic assay for phospholipase C from Bacillus cereus.
• Anal Biochem. 1995; 229: 313-6
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• A standard chromogenic assay for phosphatidylcholine determination has been modified in the development of a facile method for measuring the rate parameters (Km and Vmax) for phosphatidylcholine-preferring phospholipase C from Bacillus cereus (PLCBc). The assay is applied to the determination of the Ki for a known inhibitor of PLCBc.

• Carne A et al.
• A beta-subclass phosphatidylinositol-specific phospholipase C from squid (Loligo forbesi) photoreceptors exhibiting a truncated C-terminus.
• FEBS Lett. 1995; 372: 243-8
• Display abstract

• A PCR-based strategy has been used to isolate a full length cDNA encoding a phosphatidylinositol-specific phospholipase C from a sized cDNA squid (Loligo forbesi) retinal library. The predicted protein sequence contains 875 amino acids, with calculated M(r) 98,181, and has marked similarity with PLC beta-isoforms, including conservation of the 'X' and 'Y' regions. It is unique in having a major C-terminal truncation. A major protein of apparent M(r) 120,000 estimated by SDS-PAGE has been isolated from squid photoreceptors and identified by partial protein sequence analysis to correspond to the protein sequence predicted from the cDNA clone. This protein has been shown to hydrolyse phosphatidylinositol 4,5-bisphosphate. It is not yet clear whether this represents the major light-activated PLC in squid vision.

• Drobnik W, Mollers C, Resink T, Schmitz G
• Activation of phosphatidylinositol-specific phospholipase C in response to HDL3 and LDL is markedly reduced in cultured fibroblasts from Tangier patients.
• Arterioscler Thromb Vasc Biol. 1995; 15: 1369-77
• Display abstract

• We compared HDL3- and LDL-induced signal transduction in normal and Tangier fibroblasts to elucidate whether impaired signal transduction responses to lipoproteins might contribute to disturbed cellular lipid and lipoprotein metabolism in Tangier disease, a rare autosomal disorder of cellular lipid and lipoprotein metabolism. In several cell types HDL and LDL activate a currently unknown isoform of phosphatidylinositol-specific phospholipase C (PI-PLC) that results in the generation of 1,2-diacylglycerol and inositol 1,4,5-trisphosphate. Compared with normal fibroblasts, Tangier fibroblasts stimulated with HDL3 or LDL resulted in a significantly reduced accumulation of inositol phosphates and 1,2-diacylglycerol formation. Furthermore, in Tangier fibroblasts both lipoproteins failed to mobilize calcium from internal pools, and the cytosol-to-membrane redistribution of protein kinase C (in both the alpha and epsilon isoforms) was markedly reduced. Thus, the data indicate an impaired PI-PLC activation in response to lipoproteins in Tangier fibroblasts.

• Shortridge RD, McKay RR
• Invertebrate phosphatidylinositol-specific phospholipases C and their role in cell signaling.
• Invert Neurosci. 1995; 1: 199-206
• Display abstract

• Phosphatidylinositol-specific phospholipase C (PLC) is a family of enzymes that occupy a pivotal role in one of the largest classes of cellular signaling pathways known. Mammalian PLC enzymes have been divided into four major classes and a variety of subclasses based on their structural characteristics and immunological differences. There have been five invertebrate PLC-encoding genes cloned thus far and these fall within three of the four major classes used in categorizing mammalian PLC. Four of these invertebrate genes have been cloned from Drosophila melanogaster and one is from Artemia, a brine shrimp. Structural characteristics of the invertebrate enzymes include the presence of highly conserved Box X and Box Y domains found in major types of mammalian PLC as well as novel features. Two of the invertebrate PLC genes encode multiple splice-variant subtypes which is a newly emerging level of diversity observed in mammalian enzymes. Studies of the invertebrate PLCs have contributed to the identification of the physiological functions of individual isozymes. These identified roles include cellular processes such as phototransduction, olfaction, cell growth and differentiation.

• Henry RA, Boyce SY, Kurz T, Wolf RA
• Stimulation and binding of myocardial phospholipase C by phosphatidic acid.
• Am J Physiol. 1995; 269: 34958-34958
• Display abstract

• Exposure of adult ventricular myocytes to exogenous natural phosphatidic acid results in the production of inositol phosphates by unknown mechanism(s). We characterized stimulation of myocytic phosphoinositide-specific phospholipase C (PLC) by synthetic dioleoyl phosphatidic acid (PA) as a potential mechanism for modulation of inositol phosphate production. Our data demonstrate that exogenous PA, at 10(-8)-10(-5) M, caused a concentration-dependent increase in inositol 1,4,5-trisphosphate in adult rabbit ventricular myocytes. PA also caused a concentration-dependent increase in in vitro activity of myocytic PLC in the presence or absence of ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA). PLC-delta 1, the predominant isozyme of PLC expressed in adult rabbit ventricular myocytes, bound to liposomes of PA with high affinity in the presence of EGTA. The phosphomonoester group of PA was critical to in vitro stimulation of myocytic PLC activity and high-affinity binding of PLC-delta 1. We propose that binding of PLC-delta 1 to phosphatidic acid may be a novel mechanism for dynamic membrane association and modulation of PLC in adult ventricular myocytes.

• Shanmugasundaram GK, Munuswamy N
• Identification and partial purification of phosphatidylcholine phospholipase C from the cryptobiotic cysts of the fairy shrimp, Streptocephalus dichotomus.
• Biochem Mol Biol Int. 1995; 36: 203-7
• Display abstract

• The occurrence of phosphatidyl choline specific phospholipase C (PC-PLC)(EC 3.1.4.3) which liberates diacylglycerol (DAG), an important second messenger molecule, has been reported for the first time from the cryptobiotic cysts of the fairy shrimp, Streptocephalus dichotomus. Since the cysts undergo a revival of metabolism and resumption of development upon hydration, the possible role of the enzyme, in these processes was investigated. The results revealed enzyme's presence and an increase in its activity with the advancement of cyst development. Further partial purification of the enzyme using a lipoprotein linked- Sepharose CL-4B column (an affinity chromatographic principle) was attempted. The purification of 19.3 fold with a yield of 80% was attained.

• Cheng HF et al.
• Cloning and identification of amino acid residues of human phospholipase C delta 1 essential for catalysis.
• J Biol Chem. 1995; 270: 5495-505
• Display abstract

• In vitro single point mutagenesis, inositol phospholipid hydrolysis, and substrate protection experiments were used to identify catalytic residues of human phosphatidylinositide-specific phospholipase C delta 1 (PLC delta 1) isolated from a human aorta cDNA library. Invariant amino acid residues containing a functional side chain in the highly conserved X region were changed by in vitro mutagenesis. Most of the mutant enzymes were still able to hydrolyze inositol phospholipid with activity ranging from 10 to 100% of levels in the wild type enzyme. Exceptions were mutants with the conversion of Arg338 to Leu (R338L), Glu341 to Gly (E341G), or His356 to Leu (H356L), which made the enzyme severely defective in hydrolyzing inositol phospholipid. Phospholipid vesicle binding experiments showed that these three cleavage-defective mutant forms of PLC delta 1 could specifically bind to phosphatidylinositol 4,5-bisphosphate (PIP2) with an affinity similar to that of wild type enzyme. Western blotting analysis of trypsin-treated enzyme-PIP2 complexes revealed that a 67-kDa major protein fragment survived trypsin digestion if the wild type enzyme, E341G, or H356L mutant PLC delta 1 was preincubated with 7.5 microM PIP2, whereas if it was preincubated with 80 microM PIP2, the size of major protein surviving was comparable to that of intact enzyme. However, mutant enzyme R338L was not protected from trypsin degradation by PIP2 binding. These observations suggest that PLC delta 1 can recognize PIP2 through a high affinity and a low affinity binding site and that residues Glu341 and His356 are not involved in either high affinity or low affinity PIP2 binding but rather are essential for the Ca(2+)-dependent cleavage activity of PLC.

• Abogadie FC, Bruch RC, Wurzburger R, Margolis FL, Farbman AI
• Molecular cloning of a phosphoinositide-specific phospholipase C from catfish olfactory rosettes.
• Brain Res Mol Brain Res. 1995; 31: 10-6
• Display abstract

• A 2.7 kb clone encoding the partial (about 66%) sequence of a phosphoinositide-specific phospholipase C (PLC) was isolated from a cDNA library constructed from channel catfish (Ictalurus punctatus) olfactory rosettes. The clone, designated 30c7, was completely sequenced by automated DNA sequencing and was found to share significant homology with rat and bovine PLCs of the delta 1 isotype. In situ hybridization showed that 30c7 transcripts were expressed in a small subpopulation of olfactory neurons, as well as in other cell types in the olfactory epithelium. Polymerase chain reaction (PCR) analysis indicated that the enzyme was also expressed in several additional tissues, including brain, gill, heart, liver and skeletal muscle. These results suggest that the PLC encoded by clone 30c7 is expressed in several tissues and therefore may have a role in mediating transduction events in diverse tissues as well as in a small group of olfactory neurons.

• Lee KH, Cho YJ, Lee SB, Cho KC, Cha SH, Endou H
• Evidence suggesting a role for phospholipase C isozyme, PLC-delta 1 in corticomedullary osmotic gradients in rat kidneys.
• Biochem Mol Biol Int. 1995; 37: 25-31
• Display abstract

• We determined the distributional patterns of phospholipase C isozymes within the rat kidneys. PLC-beta1 was localized in the inner medulla at the highest degree followed by the inner stripe of the outer medulla, the cortex and the outer stripe of the outer medulla. PLC-gamma1 was distributed homogeneously along the corticomedullary axis. PLC-delta1 showed gradual increase from the cortex to the inner medulla. Tissue osmotic gradients were measured using 4 slices, resulting in gradual increase from the cortex to the inner medulla. The pattern of PLC-delta1 appeared very similar to the osmotic gradient in the kidney. The results suggest that the distinct patterns of the PLC isozymes may be associated with different signal transduction pathways along the corticomedullary axis and PLC-delta1 may play a role in the osmoregulation of the medullary region.

• Hirata M, Yagisawa H
• [Regulation of phospholipase C-delta 1]
• Seikagaku. 1995; 67: 1128-32

• Lagercrantz J et al.
• Genomic organization and complete cDNA sequence of the human phosphoinositide-specific phospholipase C beta 3 gene (PLCB3).
• Genomics. 1995; 26: 467-72
• Display abstract

• We have characterized the complete cDNA sequence, genomic structure, and expression of the human phosphoinositide-specific phospholipase C beta 3 (PLC beta 3) gene (gene symbol PLCB3). PLC beta 3 plays an important role in initiating receptor-mediated signal transduction. Activation of PLC takes place in many cells as a response to stimulation by hormones, growth factors, neurotransmitters, and other ligands. The partial cDNA sequence of PLC beta 3, previously published, was extended with 876 bp in the 5' direction, giving a transcript of 4400 bp and a total open reading frame of 1234 amino acids. This was in accordance with expression analysis by Northern blotting that revealed a single 4.4-kb transcript in all tissues tested. Genomic data were obtained by sequencing plasmid subclones of a cosmid that contained the whole gene. The size of the complete transcription unit was estimated to be on the order of 15 kb. The gene contains 31 exons, with all splice donor and acceptor sites conforming to the GT/AG rule. No exon exceeds 571 bp in length, and the shortest exon spans only 36 bp. More than half of the introns are smaller than 200 bp, with the smallest being only 79 bp long. The transcription initiation site was determined to be within an 8-bp cluster 328-321 bp upstream of the translation initiation site. The 5'flanking region is highly GC rich, with multiple CpG doublets, and contains multiple binding sites for Sp1. Lacking typical transcriptional regulatory sequences such as TATA and CAAT boxes, the putative promoter region conforms to the group of housekeeping promoters.

• Claro E, Sarri E, Picatoste F
• Measurement of phospholipase C activity in brain membranes.
• Methods Mol Biol. 1995; 41: 177-88

• Williams S, Mesaeli N, Panagia V
• Phospholipase signalling pathways in thyroxine-induced cardiac hypertrophy.
• Ann N Y Acad Sci. 1995; 752: 187-91

• Birrell GB, Hedberg KK, Griffith OH
• An extracellular inositol phospholipid-specific phospholipase C is released by cultured Swiss 3T3 cells.
• Biochem Biophys Res Commun. 1995; 211: 318-24
• Display abstract

• A phosphatidylinositol-cleaving phospholipase C (PI-PLC) activity is released into the extracellular environment by intact Swiss 3T3 cell cultures. The activity is found in both serum-containing and serum-free defined culture medium. The cells remain attached and intact by Trypan Blue exclusion and lactate dehydrogenase assays. The activity is specific for phosphoinositides as no cleavage of phosphatidylcholine is observed. The activity is a phospholipase C rather than D since the water soluble products formed from cleavage of [3H]phosphatidylinositol were inositol phosphates and not inositol. Analysis of the inositol phosphate products showed a variation in composition with the pH of the assay, the ratio of noncyclic:cyclic forms being 60:40 at pH 7.5 and 40:60 at pH 5.5. This external phospholipase C resembles the well-characterized intracellular isozymes in that it is calcium-dependent and has a pH optimum between 5 and 6. From membrane filter assays the molecular weight of the native enzyme is estimated to be between 50 and 100 kDa.

• Bertagnolo V et al.
• Identification of PI-PLC beta 1, gamma 1, and delta 1 in rat liver: subcellular distribution and relationship to inositol lipid nuclear signalling.
• Cell Signal. 1995; 7: 669-78
• Display abstract

• The subcellular distribution of PI-PLC beta 1, gamma 1, and delta 1 has been investigated in rat liver by western blot and immunohistochemical analysis with a panel of isoform-specific antibodies. The data obtained in situ on cryo-sectioned tissue indicate that PI-PLC beta 1 is predominantly nuclear, while gamma 1 is largely cytoplasmic and delta 1 is sharply restricted to the cytoplasm. In fractionation experiments, the Western blot analysis indicated that the recovery of the nuclear isoforms beta 1 and gamma 1 was not affected by the removal of the nuclear membrane, and that the two enzymes persisted in nuclear matrix and lamina, obtained after nuclease digestion and extraction with high salt and detergent. The assay of the phosphodiesterase activity in different cell fractions correlates with the observed relative abundance of the enzymes, and specific inhibition with neutralizing anti-beta 1 and -gamma 1 isoforms confirms that these are the enzymes active at the nuclear level. These results demonstrate that in rat liver cells, as in other cell types, different members of the PI-PLC family show a discrete intracellular distribution, and suggest that PI-PLC beta 1 and gamma 1 play a central role in modulating the nuclear phosphoinositide cycle.

• Simoes AP, Camps M, Schnabel P, Gierschik P
• Mutational analysis of a putative polyphosphoinositide binding site in phospholipase C-beta 2.
• FEBS Lett. 1995; 365: 155-8
• Display abstract

• The phosphatidylinositol 4,5-bisphosphate (PtdIns-P2)-regulated actin-binding protein gelsolin and most phosphoinositide-specific phospholipases C (PLCs) comprise a basic amino acid motif ((K/R)xxxKxK(K/R); x denotes any amino acid) which was previously suggested to represent a PtdInsP2-binding site commonly present in these proteins. We have challenged this hypothesis for PLC beta 2 by replacing one or several residues of this motif (KILIKNKK; residues 457-464) and examining the functional consequences of these alterations. The results show that the integrity of the basic motif is important for PtdInsP2 hydrolysis by PLC beta 2. Replacement of lysines 463 or 461 by arginine led to reduction or complete loss, respectively, of enzyme activity. The results provide further support to the concept that the function of the basic motif within the various PLCs is to bind the enzyme substrate PtdInsP2.

• Simoes AP, Reed J, Schnabel P, Camps M, Gierschik P
• Characterization of putative polyphosphoinositide binding motifs from phospholipase C beta 2.
• Biochemistry. 1995; 34: 5113-9
• Display abstract

• Several phosphatidylinositol 4,5-bisphosphate (PtdInsP2)-regulated actin-binding proteins and most phosphoinositide-specific phospholipases C (PI-PLCs) comprise a basic amino acid motif (KxxxKxKK, where x denotes any amino acid), which was previously suggested to represent a PtdInsP2-binding site commonly present in these proteins. We have shown earlier that a peptide corresponding to amino acids 448-464 of human PLC beta 2 (LPSPEDLRGKILIKNKK, peptide P1) markedly and specifically stimulated the activity of this enzyme [Simoes et al. (1993) FEBS Lett. 331, 248]. Here, we present a detailed analysis of the effects of various peptides related to peptide P1 aimed at understanding the mechanisms of peptide-mediated PLC beta 2 stimulation. Peptide KILIKNKK (P2), which comprises only the basic amino acid consensus motif, also stimulated PLC beta 2, although higher concentrations were required to observe this stimulatory effect. The effects of P1 and P2 were not additive, indicating that the two peptides affect PLC beta 2 activity via the same mechanism. Peptide LPSPEDLRG (P3), composed of the amino-terminal half of P1, did not affect the activity of PLC beta 2. Peptide KILIKNKKQFSGPTSS (P4), which includes the nine amino acids flanking the carboxy-terminus of the KILIKNKK motif within the sequence of PLC beta 2, stimulated the enzyme but was indistinguishable in potency from P2. Circular dichroism analysis revealed that peptide P1 changes its conformation in the presence of PtdInsP2 but not in the presence of other phospholipids including phosphatidylinositol 4-phosphate. The results suggest that the basic amino acid sequence physically interacts with PtdInsP2.(ABSTRACT TRUNCATED AT 250 WORDS)

• McDonald LJ, Mamrack MD
• Phosphoinositide hydrolysis by phospholipase C modulated by multivalent cations La(3+), Al(3+), neomycin, polyamines, and melittin.
• J Lipid Mediat Cell Signal. 1995; 11: 81-91
• Display abstract

• Second messenger production from phosphoinositide hydrolysis is regulated by different pathways, such as G-proteins or tyrosine phosphorylation of phosphoinositide phospholipase C (PI-PLC). Another means of altering the activity of PI-PLC is through cation interaction with the phosphoinositide substrate. A variety of organic and inorganic multi-valent cations were examined for their effects on the activity of purified PI-PLC delta. Surprisingly, the cations produced both stimulation and inhibition of PI-PLC catalyzed phosphoinositide hydrolysis, depending on the substrate and the ion to phosphoinositide stoichiometry. These data support the hypothesis that ionic complexes with phosphoinositides may alter their hydrolysis by PI-PLC.

• Shi J, Gonzales RA, Bhattacharyya MK
• Characterization of a plasma membrane-associated phosphoinositide-specific phospholipase C from soybean.
• Plant J. 1995; 8: 381-90
• Display abstract

• Phosphoinositide-specific phospholipase C (PI-PLC) is a key signal transducing enzyme which generates the second messengers inositol trisphosphate and diacylglycerol in mammalian cells. A cDNA clone (PI-PLC1) encoding a phosphoinositide-specific phospholipase C was isolated from soybean by screening a cDNA expression library using an anti-(plasma membrane) serum. Genomic DNA gel blot analysis suggested that the corresponding gene is a member of a multigene family. The deduced amino acid sequence of the soybean PI-PLC1 isozyme contains the conserved X and Y regions, found in other PI-PLCs. It is closely related to mammalian delta-type PI-PLCs, Dictyostelium discoideum PI-PLC and yeast PI-PLC1 in terms of the arrangement of the conserved region. Unlike mammalian delta-type PI-PLCs and yeast PI-PLC1, the putative Ca(2+)-binding site of the soybean PI-PLC1 is located in the region spanning the X and Y domains, and the N-terminal region is truncated. FLAG epitope-tagged PI-PLC1 fusion protein purified from transgenic tobacco plants showed phosphoinositide-specific phospholipase C activity. Heterologous expression of the soybean PI-PLC1 cDNA in a yeast PI-PLC1 deletion mutant complemented the lethality phenotype of haploid PI-PLC1 disruptants. Immunoblot analysis of the cell fractions prepared from transgenic tobacco plants over-expressing the FLAG epitope-tagged PI-PLC1 fusion protein indicated that the protein encoded by the PI-PLC1 cDNA was localized in the cytosol and plasma membrane.

• Ui H, Imoto M, Umezawa K
• Inhibition of phosphatidylinositol-specific phospholipase C activity by fluvirucin B2.
• J Antibiot (Tokyo). 1995; 48: 387-90
• Display abstract

• We isolated fluvirucin B2 from the culture broth of Streptomyces as an inhibitor of phosphatidylinositol-specific phospholipase C (PI-PLC). It inhibited PI-PLC of A431 cell cytosol with an IC50 of 1.6 micrograms/ml. Fluvirucin B2 also inhibited PI-PLC in cultured A431 cells, whereas it did not inhibit phosphatidylinositol synthesis and macromolecular synthesis markedly. It also inhibited epidermal growth factor-induced rapid rounding of A431 cells, in which PI turnover is involved.

• Yagisawa H et al.
• Expression and characterization of an inositol 1,4,5-trisphosphate binding domain of phosphatidylinositol-specific phospholipase C-delta 1.
• J Biol Chem. 1994; 269: 20179-88
• Display abstract

• It was previously found that the 85-kDa protein purified from rat brain using an inositol 1,4,5-trisphosphate (Ins(1,4,5)P3)-immobilized matrix was the delta 1 isoform of phosphatidylinositol-specific phospholipase C (PLC). We expressed rat PLC-delta 1 in Escherichia coli as a fusion protein with glutathione S-transferase, and found that the bacterial lysate shows a significant amount of Ins(1,4,5)P3 binding. The lysate was applied to Ins(1,4,5)P3-immobilized column chromatography and the eluate with 2 M NaCl solution containing only a 100-kDa protein showed high Ins(1,4,5)P3 binding. The lysate was also purified to near homogeneity using a glutathione-Sepharose 4B affinity system. Bacterially-expressed enzyme thus purified showed essentially the same inositol phosphate binding characteristics as the brain-derived enzyme. PLC-delta 1 consists of the amino-terminal nonconserved region and two well-conserved regions among isozymes, designated as X and Y, which are thought to constitute a catalytic core of the enzyme. Using a combination of deletion mutants and proteolytic products of the enzyme, we were able to locate an Ins(1,4,5)P3 binding domain in the molecule. Deletion of 223 residues from the amino terminus completely abolished the binding activity, while deletion of X region only partially inhibited the binding and deletion of Y region did not affect the binding. A 76-kDa proteolytic product of the expressed PLC-delta 1 which lacked 60 amino acids at the amino terminus showed a minimal Ins(1,4,5)P3 binding activity. A peptide consists of 14 amino acids corresponding to residues 30-43 of PLC-delta 1, which contains 6 basic amino acids, binds to an Ins(1,4,5)P3-immobilized matrix. Moreover, Ins(1,4,5)P3 binding was blocked by phospholipid vesicles containing phosphatidylinositol 4,5-bisphosphate. These results, taken together, indicate that the amino-terminal domain of PLC-delta 1 is important for the binding of both Ins(1,4,5)P3 and phosphatidylinositol 4,5-bisphosphate.

• Yorek MA, Dunlap JA, Stefani MR, Davidson EP, Zhu X, Eichberg J
• Decreased myo-inositol uptake is associated with reduced bradykinin-stimulated phosphatidylinositol synthesis and diacylglycerol content in cultured neuroblastoma cells exposed to L-fucose.
• J Neurochem. 1994; 62: 147-58
• Display abstract

• L-Fucose is a potent, competitive inhibitor of myo-inositol transport by cultured mammalian cells. Chronic exposure of neuroblastoma cells to L-fucose causes a concentration-dependent decrease in myo-inositol content, accumulation, and incorporation into phosphoinositides. In these studies, L-fucose supplementation of culture medium was used to assess the effect of decreased myo-inositol metabolism and content on bradykinin-stimulated phosphatidylinositol synthesis and diacylglycerol production. Chronic exposure of cells to 30 mM L-fucose caused a sustained decrease in bradykinin-stimulated, but not basal, 3H-inositol phosphate release and 32P incorporation into phosphatidylinositol in cells incubated in serum-free, unsupplemented medium. In addition, 32P incorporation into phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate was not altered in L-fucose-conditioned cells. Acute exposure of cells to serum-free medium containing 30 mM L-fucose did not affect either basal or bradykinin-stimulated 32P incorporation into phosphatidylinositol. Basal diacylglycerol content was decreased by 20% in cells chronically exposed to 30 mM L-fucose, although analysis of the molecular species profile revealed no compositional change. Bradykinin stimulated diacylglycerol production in neuroblastoma cells by increasing the hydrolysis of both phosphoinositides and phosphatidylcholine. Bradykinin-stimulated production of total diacylglycerol was similar for control and L-fucose-conditioned cells. However, there was a decrease in the bradykinin-induced generation of the 1-stearoyl-2-arachidonoyl diacylglycerol molecular species in the cells chronically exposed to 30 mM L-fucose. This molecular species accounts for about 70% of the composition of phosphoinositides, but only 10% of phosphatidylcholine. The results suggest that a decrease in myo-inositol uptake results in diminished agonist-induced phosphatidylinositol synthesis and phosphoinositide hydrolysis in cultured neuroblastoma cells grown in L-fucose-containing medium.

• Momchilova-Pankova AB, Markovska TT, Yanev EI, Koumanov KS
• Phospholipase C activities in rat liver plasma membranes depend on the phospholipid composition.
• J Lipid Mediat Cell Signal. 1994; 9: 235-46
• Display abstract

• Investigations were carried out on the influence of rat liver plasma membranes phospholipid composition on phospholipase C activity using PIP, PIP2, PC and PE as substrates. The membrane phospholipids were modified by incorporation of definite phospholipids with the aid of lipid transfer proteins or after partial delipidation with exogenous phospholipases A2 and C. The results indicated that sphingomyelin inhibited all phospholipase C activities. The incorporation of two different molecular species of phosphatidylcholine did not alter significantly the investigated phospholipase C activities, indicating that membrane fluidity was not essential in this case. Phosphatidylglycerol, phosphatidylserine, phosphatidylinositol and phosphatidylethanolamine served as specific activators of plasma membrane-bound phospholipase C when PIP, PIP2 and PC were used as substrates. However, these four phospholipids inhibited phospholipase C activity towards PE. The role of phosphoinositide-specific phospholipase C in the production of second messengers as well as the eventual biological significance of PC and PE as substrates for phospholipase C is discussed.

• Volwerk JJ, Filthuth E, Griffith OH, Jain MK
• Phosphatidylinositol-specific phospholipase C from Bacillus cereus at the lipid-water interface: interfacial binding, catalysis, and activation.
• Biochemistry. 1994; 33: 3464-74
• Display abstract

• Binding characteristics of phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus cereus binding to the phospholipid-water interface were determined by spectroscopic methods and correlated with PI-PLC's catalytic properties. Binding of the enzyme to micelles and bilayers of zwitterionic phosphocholines is accompanied by an increase in the fluorescence emission from tryptophan, whereas a decrease in the emission is observed with synthetic anionic lipids containing a phosphomethanol head group. A similar decrease in the tryptophan emission is observed with phosphatidylinositol (PI) analogues containing the phospho-D-1-myo-inositol head group, but not with the enantiomeric L-1-myo-inositol. In covesicles of PI and phosphatidylcholine (PC), the rate of cleavage of PI is reduced because, as a neutral diluent, PC effectively reduces the surface concentration of PI that the bound enzyme "sees" in the interface. This permits determination of the interfacial Michaelis constant (KM*) as 0.26 mol fraction for PI as substrate. On the other hand, ditetradecylglycerophosphomethanol (DTPM) acts as a kinetic competitive inhibitor in the covesicles. The spectroscopic and catalytic activity data taken together show that PI-PLC binds to the interface of aqueous dispersions of phospholipids with an apparent Kd (in terms of the lipid monomers) of about 10-50 microM. However, only lipids with an anionic head group, such as phosphomethanol and phospho-D-1-myo-inositol, are able to bind as single molecules into the active site of the enzyme at the interface. Enantiomeric phospho-L-1-myo-inositol or the zwitterionic phosphocholine head group has little affinity for the enzyme at the interface. Thus, PI-PLC appears to obey the two-stage, Michaelis-Menten adaptation of interfacial catalysis, according to which the binding of the enzyme to the interface precedes the steps of the catalytic turnover at the interface. Limit estimates suggest that on PI or PI/PC vesicles the catalysis occurs in the "scooting" mode with a moderate processivity. DTPM vesicles also inhibit the activity of PI-PLC toward the synthetic water-soluble substrate myo-inositol 1-(4-nitrophenyl phosphate), but the activity is enhanced severalfold in the presence of vesicles of zwitterionic phosphatidylcholine. Several possible explanations of this interfacial activation are considered within the general context of the kinetic scheme for interfacial catalysis. The kinetic results for the action of PI-PLC bound to vesicles are consistent with a model in which the interface acts as an "allosteric" effector of the catalytic rate constant, kcat, without affecting the substrate binding.

• Drayer AL, Van der Kaay J, Mayr GW, Van Haastert PJ
• Role of phospholipase C in Dictyostelium: formation of inositol 1,4,5-trisphosphate and normal development in cells lacking phospholipase C activity.
• EMBO J. 1994; 13: 1601-9
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• The micro-organism Dictyostelium uses extracellular cAMP to induce chemotaxis and cell differentiation. Signals are transduced via surface receptors, which activate G proteins, to effector enzymes. The deduced protein sequence of Dictyostelium discoideum phosphatidylinositol-specific phospholipase C (PLC) shows strong homology with the mammalian PLC-delta isoforms. To study the role of PLC in Dictyostelium, a plc- mutant was constructed by disruption of the PLC gene. No basal or stimulated PLC activity could be measured during the whole developmental programme of the plc- cells. Loss of PLC activity did not result in a visible alteration of growth or development. Further analysis showed that developmental gene regulation, cAMP-mediated chemotaxis and activation of guanylyl and adenylyl cyclase were normal. Although the cells lack PLC activity, inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] was present at only slightly lower concentrations compared with control cells. Mass analysis of inositol phosphates demonstrated the presence of a broad spectrum of inositol phosphates in Dictyostelium, which was unaltered in the plc- mutant. Cell labelling experiments with [3H]inositol indicated that [3H]Ins(1,4,5)P3 was formed in a different manner in the mutant than in control cells.

• McKay RR, Zhu L, Shortridge RD
• Membrane association of phospholipase C encoded by the norpA gene of Drosophila melanogaster.
• Neuroscience. 1994; 61: 141-8
• Display abstract

• Severe mutations within the norpA gene of Drosophila abolish the photoreceptor potential and render the fly blind by deleting phospholipase C, an essential component of the phototransduction pathway. To study the membrane association of phospholipase C, we have utilized biochemical assays of phospholipase C activity, which predominant measurable phospholipase C activity in head homogenates has been shown to be encoded by norpA, as well as antisera generated against the major gene product of norpA to examine its subcellular distribution before and during phototransduction. We find that both phospholipase C activity and the norpA protein are predominantly associated with membrane fractions in heads of both light- and dark-adapted flies. Moreover, phospholipase C activity as well as norpA protein can be easily extracted from membrane preparations of light- or dark-adapted flies using high salt, indicating that the norpA protein is peripherally localized on the membrane. These data suggest that the norpA encoded phospholipase C of Drosophila is a permanent peripheral membrane protein. If this is indeed the case, then it would mean that the reversible redistribution of phospholipase C from the cytosol to the membrane, as observed in epidermal growth factor receptor stimulation of mammalian phospholipase C gamma, is not a universal mechanism utilized by all types of phosphatidylinositol-specific phospholipase C.

• Sundell S, Hansen S, Hough E
• A proposal for the catalytic mechanism in phospholipase C based on interaction energy and distance geometry calculations.
• Protein Eng. 1994; 7: 571-7
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• The non-specific phospholipase C from Bacillus cereus preferentially hydrolyses phosphatidylcholine but is also active against phosphatidylserine, phosphatidylethanolamine and at a much lower level, sphingomyelin. A minimal substrate model containing all required structural and configurational elements of a high affinity substrate was docked into the active site. The enzyme-substrate attachment points were from molecular interaction energy calculations using the program GRID and from a previous phosphate inhibitor complex structure. Available conformational space for the substrate was sampled by distance geometry calculations using the program DGEOM. This investigation clearly identifies the attacking nucleophile, a catalytically favourable orientation of the phosphate group in its tetra-, as well as its penta-, coordinated state and a crucial stabilizing environment for the alkoxide intermediate. Based on this information a complete catalytic cycle is proposed.

• Holler T, Klein J, Loffelholz K
• Phospholipase C and phospholipase D are independently activated in rat hippocampal slices.
• Biochem Pharmacol. 1994; 47: 411-4
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• In order to investigate a possible G-protein-mediated activation of phospholipase D (PLD) and its relationship to the activation of phosphoinositide-specific phospholipase C (PI-PLC), we measured the effects of aluminium fluoride and carbachol on choline release, the PLD-specific transphosphatidylation reaction (generation of phosphatidylpropanol) and the formation of inositol phosphates in rat hippocampal slices. Aluminium fluoride markedly enhanced the formation of choline and phosphatidylpropanol but failed to increase the formation of inositol phosphates. In contrast, the muscarinic agonist carbachol strongly stimulated PI-PLC but failed to activate PLD. We conclude that PLD in hippocampal slices is activated by a G-protein independently of phosphoinositide hydrolysis.

• Boguslavsky V, Rebecchi M, Morris AJ, Jhon DY, Rhee SG, McLaughlin S
• Effect of monolayer surface pressure on the activities of phosphoinositide-specific phospholipase C-beta 1, -gamma 1, and -delta 1.
• Biochemistry. 1994; 33: 3032-7
• Display abstract

• Three isoforms of phospholipase C, either PLC-beta 1, PLC-gamma 1, or PLC-delta 1, were added to the aqueous subphase beneath phospholipid monolayers formed at an air-solution interface, and the initial rate of hydrolysis of phosphatidylinositol 4,5-bisphosphate was measured after addition of 10 microM free Ca2+. The monolayers were formed from mixtures of phosphatidylcholine (65% PC), phosphatidylserine (33% PS), and phosphatidylinositol 4,5-biphosphate (2% PIP2). Increasing the surface pressure of the monolayer, pi, from 15 to 25 mN/m decreases the rate of hydrolysis 16-, 13-, and 5-fold for PLC-beta 1, PLC-gamma 1, and PLC-delta 1, respectively. The simplest interpretation of these results is that a portion of each of the enzymes of area Ap must insert into the monolayer, doing work pi Ap, prior to hydrolysis of PIP2; binding studies with simple model compounds of known cross-sectional area are consistent with this interpretation. Removing the monovalent acidic lipid PS from the monolayer decreases the initial rates of hydrolysis of PIP2 about 3-fold for each PLC isoform, which suggests that negative electrostatic surface potentials increase the PLC activity.

• Rossi MA, Di Mauro C, Esterbauer H, Fidale F, Dianzani MU
• Activation of phosphoinositide-specific phospholipase C of rat neutrophils by the chemotactic aldehydes 4-hydroxy-2,3-trans-nonenal and 4-hydroxy-2,3-trans-octenal.
• Cell Biochem Funct. 1994; 12: 275-80
• Display abstract

• A comparison has been made between the effects of 4-hydroxy-2,3-trans-nonenal (HNE) and 4-hydroxy-2,3-trans-octenal (HOE), two lipid peroxidation products, on the basal and GTPgammaS-stimulated activities of phosphoinositide-specific phospholipase C (PL-C) of rat polymorphonuclear leukocytes. PL-C activity was determined in vitro by measuring the hydrolysis of [3H] phosphatidylinositol-4,5-bis- phosphate (PtdIns-P2) added as exogenous substrate to neutrophil plasma membranes. PL-C was activated by concentrations of HNE ranging from 10(-8) to 10(-6) M both in the presence and in the absence of 2 x 10(-5) M GTPgammaS; HOE stimulated the enzymatic activity between 10(-11) and 10(-8) M; maximal stimulation was given by 10(-11) M HOE plus GTPgammaS. The aldehyde concentrations able to accelerate PtdIns-P2 breakdown displayed a good correspondence with those which have been reported to stimulate the oriented migration of rat neutrophils. Pretreatment of neutrophils with pertussis toxin prevented the stimulation of PL-C by 10(-11) M HOE and by HOE plus GTPgammaS. Our results suggest that the chemotactic action of HNE and HOE might depend on the activation of PL-C; furthermore a regulatory G protein appears to be involved in the acceleration of PtdIns-P2 turnover by HOE.

• McKay RR, Miller K, Weckstrom M, Torkkeli P, Jarvilehto M, Shortridge RD
• The rpa (receptor potential absent) visual mutant of the blowfly (Calliphora erythrocephala) is deficient in phospholipase C in the eye.
• J Neurogenet. 1994; 9: 177-87
• Display abstract

• The rpa (receptor potential absent) mutation of the blowfly, Calliphora erythrocephala, reduces the light-evoked responses of photoreceptor cells and renders the fly blind. This phenotype is similar to the phenotype caused by norpA mutations in Drosophila which have been shown to occur within a gene encoding phospholipase C. In Western blots, norpA antiserum stains a protein in homogenates of wild-type Calliphora eye and head that is similar in molecular weight to the norpA protein. Very little staining of this protein is observed in similar homogenates of rpa mutant. Moreover, norpA antiserum strongly stains retina in immunohistochemical assays of wild-type adult head, but not in rpa mutant. Furthermore, eyes of rpa mutant have a reduced amount of phospholipase C activity compared to eye of wild-type Calliphora. These data suggest that the rpa mutation occurs in a phospholipase C gene of the blowfly that is homologous to the norpA gene of Drosophila.

• Ferreira PA, Pak WL
• Bovine phospholipase C highly homologous to the norpA protein of Drosophila is expressed specifically in cones.
• J Biol Chem. 1994; 269: 3129-31
• Display abstract

• The Drosophila norpA gene encodes a phosphatidylinositol-specific phospholipase C (PI-PLC) expressed predominantly in photoreceptors and involved in phototransduction. However, no direct role for a phospholipase C in vertebrate phototransduction has been identified to date. Recently, we reported the isolation and characterization of bovine cDNAs encoding PI-PLC isoforms expressed predominantly in the retina and with higher homology to the NorpA protein than to any other known PI-PLC. Here, we present evidence that the norpA-homologous bovine retinal PI-PLCs, although found in other retinal neurons as well, are found in cones but not in rods. The results suggest that the phototransduction cascade in cones may utilize phospholipase C in addition to phosphodiesterase.

• Tompkins TA, Moscarello MA
• The mechanism of stimulation of brain phospholipase C-alpha by myelin basic protein involves specific interactions.
• Biochim Biophys Acta. 1994; 1206: 208-14
• Display abstract

• The modulation of a brain phosphoinositide-specific phospholipase C-alpha activity was studied using a variety of compounds of different charge. Detergents such as sodium deoxycholate and cetyltrimethylammonium bromide stimulated the phospholipase C activity when used alone but when used together the effects were not additive. Spermine was an effective inhibitor of the enzyme activity while the cationic peptide, Melittin, had no effect. The inositol phosphates produced by hydrolysis with phosphoinositide-specific phospholipase C were inhibitory while diacylglycerol and inositol did not affect the phospholipase activity. Myelin basic protein, which was previously shown to stimulate phospholipase C activity by 2.5-fold, did not interact with the anionic inositol phosphatases to any significant extent. Thus we concluded that the mechanism of stimulation was not due to relief of product inhibition. Crosslinking studies with the photoactivatable reagent, N-hydroxysuccinimidyl-4-azidosalicylic acid, showed that peptide 24-33 of myelin basic protein, which stimulated the activity almost as much as the native protein, interacted specifically with the phospholipase C. Thus the mechanism by which myelin basic protein stimulated the enzyme appeared to be through specific protein-protein interaction.

• Rack M, Xhonneux-Cremers B, Schraermeyer U, Stieve H
• On the Ca(2+)-dependence of inositol-phospholipid-specific phospholipase C of microvillar photoreceptors from Sepia officinalis.
• Exp Eye Res. 1994; 58: 659-64
• Display abstract

• The breakdown of phosphatidylinositol 4,5-bisphosphate (PIP2) by the action of a phosphoinositide-specific phospholipase C is a key event in the signal transduction mechanism of microvillar photoreceptors. The enzymatic activity of phospholipase C from photoreceptor cell membranes of the cuttlefish Sepia officinalis was measured at different concentrations of free calcium. A bell-shaped curve with a maximum of enzyme activity at about 0.2 to 0.7 microM of free calcium was obtained. In the range of 10 to 90 microM free Ca2+, the activity of the phospholipase C decreased to about 30%. The activity of the phospholipase was also modulated by strontium ions. The presence of 10 microM Sr2+ strongly reduced the dependence of the phospholipase from a given calcium concentration. Similarly to 10 mM EGTA, the presence of 10 microM La3+ reduced the enzymatic activity more than ten-fold. A comparison of these values with the changes of the calcium concentration measured in ventral photoreceptor cells of Limulus polyphemus in the dark (approximately 0.5 microM Ca2+) and under illumination (up to 30-100 microM Ca2+) indicates that in microvillar photoreceptor cells an elevation of the calcium concentration in the physiological range reduces the activity of the phospholipase C. The IP3 produced by the action of phospholipase C is known to elevate the intracellular Ca2+ concentration transiently, which in turn will be able to reduce the further IP3 formation. A negative feedback in this early part of the enzyme cascade of vision in invertebrates may substantially contribute to the process of adaptation.

• Alisi MA et al.
• Synthesis of acylphosphonyl analogues of phosphatidyl-myo-inositol as inhibitors of phosphatidylinositol-specific phospholipase C.
• Carbohydr Res. 1994; 265: 139-43

• Manor E, Carbonetti NH, Silverman DJ
• Rickettsia rickettsii has proteins with cross-reacting epitopes to eukaryotic phospholipase A2 and phospholipase C.
• Microb Pathog. 1994; 17: 99-109
• Display abstract

• The entry, and possibly the exit, of rickettsiae from eukaryotic cells, as well as erythrocyte lysis by some members of this group of organisms, is thought to be mediated by a phospholipase A activity even though the enzyme has not been isolated from these organisms. Evidence for phospholipase C, on the other hand, has not been reported for the genus Rickettsia. In this study, in a preliminary attempt to demonstrate the presence of phospholipase A2 and phospholipase C in the virulent Sheila Smith strain of Rickettsia rickettsii, we performed immunoblotting and immuno-gold electron microscopy using anti-phospholipase A2 and anti-phospholipase C IgG antibodies (raised against mammalian enzymes). We provide evidence for cross-reactivity of the antibodies with proteins present in R. rickettsii. Western blots showed a higher staining intensity with anti-phospholipase C antibody than with anti-phospholipase A2. According to the results obtained with the immuno-gold labeling of phospholipase A2 and phospholipase C reactive epitopes, most of the phospholipase A2 cross-reactive material appears to be associated with the membrane of the organism while the phospholipase C cross-reactive material appears to be randomly distributed throughout the cell.

• Cifuentes ME, Delaney T, Rebecchi MJ
• D-myo-inositol 1,4,5-trisphosphate inhibits binding of phospholipase C-delta 1 to bilayer membranes.
• J Biol Chem. 1994; 269: 1945-8
• Display abstract

• The binding of phosphoinositide-specific phospholipase C-delta 1 (PLC-delta 1) to bilayer membranes composed of phosphatidylcholine (PC) and phosphatidylinositol 4,5-bisphosphate (PIP2) was measured in the presence or absence of inositol phosphates. Binding was inhibited by the natural D-isomer of myo-inositol 1,4,5-trisphosphate (D-InsP3), but not by the L-isomer. The concentration of D-InsP3 required to decrease binding by 50% was 5.4 +/- 0.5 microM. 1-(alpha-Glycerophosphoryl)-D-myo-inositol 4,5-bisphosphate and D-myo-inositol 2,4,5-trisphosphate were nearly as effective as D-Ins(1,4,5)P3. D-myo-inositol monophosphate with phosphate esterified at either positions 1 or 2 of the myo-inositol ring, had no significant effect on binding. D-myo-inositol 1,4-bisphosphate weakly inhibited the binding, whereas the 4,5-isomer was nearly as potent as D-InsP3. Neither ATP nor inorganic phosphate significantly affected binding. As expected, D-Ins(1,4,5)P3 but not L-Ins(1,4,5)P3 decreased the initial rate of PIP2 hydrolysis in bilayer vesicles. The concentration required to decrease hydrolysis by 50% was 12.4 +/- 0.5 microM. A catalytic fragment of PLC-delta 1 that lacks a domain necessary for high affinity PIP2 binding was prepared as previously described (Cifuentes, M. E., Honkanen, L., and Rebecchi, M. J. (1993) J. Biol. Chem. 268, 11586-11593). In contrast to the native enzyme, the rate of PIP2 hydrolysis, catalyzed by the fragment, was not affected by D-Ins(1,4,5)P3. These data suggest that high affinity binding of the enzyme to PIP2 and processive catalysis, involve specific recognition of the 4- and 5-position phosphates of the inositol ring. Our results are consistent with feedback inhibition by the polar head group product, D-Ins(1,4,5)P3, at a step that precedes catalysis, namely interfacial recognition.

• Hirata M et al.
• D-myo-inositol 1,4,5-trisphosphate binding domain of phospholipase C-delta 1.
• Biochem Biophys Res Commun. 1994; 205: 1563-71
• Display abstract

• D-myo-Inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) binding domain of phospholipase C-delta 1 (PLC-delta 1) was determined by examining binding activity of the synthetic peptide corresponding to residues 30-43 of PLC-delta 1. The peptide coupled with carrier proteins such as keyhole limpet hemocyanin or bovine serum albumin bound Ins(1,4,5)P3, whereas a scrambled peptide with the same amino acids did not do so. Polyclonal antibody against the peptide was examined to determine whether it would cause inhibition of the Ins(1,4,5)P3 binding to PLC-delta 1. Fab fragment of antibody to the peptide did inhibit binding to PLC-delta 1, in a dose-dependent manner. Thus it seems likely that the region of residues 30-43 of PLC-delta 1 is responsible for the binding of Ins(1,4,5)P3.

• Bruzik KS, Tsai MD
• Toward the mechanism of phosphoinositide-specific phospholipases C.
• Bioorg Med Chem. 1994; 2: 49-72

• Wu CY, Chen CF, Chiang CF
• Purification of phospholipase C from rat cerebral cortex.
• Prep Biochem. 1994; 24: 99-111
• Display abstract

• Phospholipase C from rat cerebral cortex was purified to homogeneity by use of DEAE Bio-Gel A agarose, hydroxyapatite, and heparin agarose chromatography. The purified phospholipase C (PLC) was purified 622.4-fold and its molecular weight is estimated to be 97,500. We obtained a final specific activity of 3.112 mumol of phosphatidylinositol hydrolyzed/min/mg of protein. It is specific for inositol phospholipids. The purified enzyme has an apparent optimum pH 7.0. Calcium is required for its activity. Western blotting analysis showed that two proteins were recognized by anti-PLC antiserum.

• Bruzik KS, Hakeem AA, Tsai MD
• Are D- and L-chiro-phosphoinositides substrates of phosphatidylinositol-specific phospholipase C?
• Biochemistry. 1994; 33: 8367-74
• Display abstract

• Derivatives of chiro-inositol have been recently shown to mediate many important biological processes. This work addresses the question of whether phosphatidylinositol-specific phospholipase C (PI-PLC) could be involved in the generation of these chiro-inositol derivatives. Two diastereomers of the analog of phosphatidylinositol containing 1D- and 1L-chiro-inositol have been synthesized. 1D-2-O-(1,2-O-Dipalmitoyl-sn-glycero-3-phospho)-chiro-inositol (1D-chiro-PI) was synthesized in 12 steps starting from 1D-2,3,4,5-O-tetrakis(methoxymethylene)-myo-inositol by the inversion of the hydroxyl group at the 1-position of inositol followed by several protection/deprotection and phosphorylation steps. IL-2-O-(1,2-O-Dipalmitoyl-sn-glycero-3-phospho)-chiro-inositol (1L-chiro-PI) was synthesized in eight steps starting from 1L-chiro-inositol using regioselective silylation of the hydroxyl group at the 2-position of chiro-inositol in a key synthetic stage. Both diastereomers were subjected to cleavage by PI-PLC from Bacillus thuringiensis. The reaction of 1L-chiro-PI produced chiro-inositol 1,2-cyclic phosphate, however, at the rate of 10(-3) of that attained with the natural substrate, phosphatidylinositol. On the other hand, 1D-chiro-PI was found to be resistant to PI-PLC. These results suggest that the natural chiro-inositol derivatives should have the 1L-configuration if they are produced by PI-PLC, which is in contrast to the 1D-configuration reported by others. We therefore have isolated chiro-inositol from the total bovine liver lipid and determined its absolute configuration. The obtained chiro-inositol was found to be exclusively of the 1L-configuration, with the enantiomeric purity exceeding 99%.

• Homma Y
• [Structure and function of inositol phospholipid-specific phospholipase C]
• Seikagaku. 1994; 66: 1091-110

• Tsutsumi T, Kobayashi T, Ueda H, Yamauchi E, Watanabe S, Okuyama H
• Lysophosphoinositide-specific phospholipase C in rat brain synaptic plasma membranes.
• Neurochem Res. 1994; 19: 399-406
• Display abstract

• A membrane preparation from rat brain catalyzed the hydrolysis of [2-3H]glycerol-labeled lysophosphatidylinositol (lysoPI) to yield monoacylglycerol (MG) and inositolphosphates. This phospholipase C activity had an optimal pH of 8.2. The membrane preparation did not require the addition of Ca2+ for its maximum activity, but the activity was inhibited by addition of 0.1 mM EDTA to the assay mixture and was restored by simultaneous addition of 0.2 mM Ca2+. The activity was found to be localized in synaptic plasma membranes prepared by Ficoll and Percoll density gradients. The phospholipase C was highly specific for lysoPI; diacylglycerol formation from phosphatidylinositol, and MG formation from lysophosphatidylcholine, lysophosphatidylethanolamine, and lysophosphatidylserine were below 5% of that observed with lysoPI under the conditions used. We concluded that there is a pathway for phosphatidylinositol metabolism in brain synaptic membranes which is different from the well-characterized phosphoinositide-specific phospholipase C pathway.

• Banno Y, Okano Y, Nozawa Y
• Thrombin-mediated phosphoinositide hydrolysis in Chinese hamster ovary cells overexpressing phospholipase C-delta 1.
• J Biol Chem. 1994; 269: 15846-52
• Display abstract

• The regulatory mechanism(s) of a phosphoinositide-specific phospholipase C, PLC-delta 1, was investigated using a clone of stably overexpressed PLC-delta 1 (PLC delta 30 cells) in Chinese hamster ovary cells. Thrombin stimulation of PLC delta 30 cells exhibited 6.5-fold increase in total inositol phosphates (InsP), which was significantly higher than that in the vector-transfected (V1) cells (2.0-fold). AIF-4 increased InsP accumulation in both V1 and PLC delta 30 cells, and pertussis toxin partially blocked InsP accumulation in thrombin-stimulated PLC delta 30 cells. Guanosine thiotriphosphate (GTP gamma S) markedly potentiated thrombin-stimulated InsP generation in permeabilized PLC delta 30 cells compared with V1 cells, suggesting possible involvement of a G-protein (s) in the activation of PLC-delta 1. In PLC delta 30 cells, ionomycin-induced significant InsP generation and thrombin-stimulated InsP generation were completely inhibited by addition of EGTA. Furthermore, the stimulatory effects of thrombin plus GTP gamma S in PLC delta 30 cells were more sensitive to change in free calcium concentration than in V1 cells. Suppression by 12-O-tetradecanoylphorbol 13-acetate of thrombin-stimulated InsP accumulation was not affected by increasing Ca2+ concentration. These results indicate that thrombin-induced PLC-delta 1 activation is regulated via both G-protein(s) and calcium.

• Kim CK, Park KM
• Liposome immunoassay (LIA) for gentamicin using phospholipase C.
• J Immunol Methods. 1994; 170: 225-31
• Display abstract

• A simple and sensitive liposome immunoassay for gentamicin was developed using the cytolytic agent, phospholipase C, instead of complement. Liposomes entrapping a fluorescent marker, calcein, were prepared by the reverse-phase evaporation method from a mixture of dimyristoylphosphatidylcholine and cholesterol (molar ratio of 3:1). Gentamicin, a model analyte, was covalently coupled to phospholipase C by 1-ethyl-3-(3-dimethylamino-propyl)-carbodiimide and N-hydroxysuccinimide. Liposomes were lysed by gentamicin-phospholipase C conjugate and an entrapped fluorescent marker was released. The lytic activity of gentamicin-phospholipase C conjugate was inhibited in the presence of gentamicin antiserum. The standard calibration curve was constructed by plotting percentage of liposome lysis versus log concentration of free gentamicin. The standard calibration curve was linear over 2.5 pg/ml approximately 2.5 ng/ml of gentamicin concentrations. This newly developed immunoassay is simple, relatively rapid and potentially applicable to the determination of concentration of antigens, drugs and endogenous substances.

• Butikofer P, Brodbeck U
• Partial purification and characterization of a (glycosyl) inositol phospholipid-specific phospholipase C from peanut.
• J Biol Chem. 1993; 268: 17794-802
• Display abstract

• We have isolated a glycosyl inositol phospholipid (GIP) anchor-hydrolyzing activity from peanut seeds by a series of column chromatographic steps. The activity has a pH optimum below 6.0, requires calcium, and is inhibited by sulfhydryl reagents. It cleaves the GIP anchors of solubilized acetylcholinesterase from bovine erythrocytes and variant surface glycoprotein from Trypanosoma brucei. On the other hand, it does not act on membrane-bound GIP-anchored substrate or on inositol-acylated GIP anchor of human erythrocyte acetylcholinesterase. The only product released from [3H]myristate-labeled variant surface glycoprotein following treatment with the activity from peanut was 3H-labeled diacylglycerol. Together, these findings identify the activity from peanut seeds as a GIP anchor-hydrolyzing phospholipase C. The enzyme has been found to hydrolyze not only protein GIP anchors but also phosphatidylinositol, whereas it shows no activity against other phospholipids. The water-soluble products of phosphatidylinositol hydrolysis by peanut phospholipase C were characterized as a mixture of inositol 1,2-cyclic phosphate and inositol phosphate.

• Cifuentes ME, Honkanen L, Rebecchi MJ
• Proteolytic fragments of phosphoinositide-specific phospholipase C-delta 1. Catalytic and membrane binding properties.
• J Biol Chem. 1993; 268: 11586-93
• Display abstract

• Active proteolytic fragments of phosphoinositide-specific phospholipase C-delta 1 (PLC-delta 1) were generated by trypsin digestion of the native protein. Brief proteolysis produced a 77-kDa fragment that contained the highly conserved X and Y regions but lacked the amino-terminal domain (amino acids 1-60). Prolonged digestion of PLC-delta 1 produced two fragments, one of 45 kDa that contained the entire X region and another of 32 kDa that consisted of the entire Y region and COOH-terminal domain. The 45- and 32-kDa fragments were isolated as an active heterodimeric complex. The 77-kDa fragment and the complex catalyzed calcium-dependent hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) in detergent/phospholipid mixed micelles. When compared with the native enzyme, both the 77-kDa fragment and the complex exhibited a reduced capacity to processively hydrolyze PIP2; increasing the mole fraction of PIP2 in the mixed micelle surface greatly increased the rate of PIP2 hydrolysis catalyzed by the native enzyme but not the fragments. Both fragments also exhibited a reduced affinity for substrate; the native enzyme bound to bilayer vesicles consisting of phosphatidylcholine and PIP2 with high affinity (Ka approximately 10(6) M-1), whereas the fragments bound weakly (Ka < 10(4) M-1). These results demonstrate that the X, Y, and COOH-terminal regions form a calcium-dependent catalytic core that is resistant to proteolysis. The amino-terminal domain appears to be essential for high affinity binding to PIP2 but not catalysis. These observations are consistent with the idea that the amino-terminal domain forms part of a PIP2 binding site, which anchors PLC-delta 1 to the membrane surface during processive hydrolysis of its substrate.

• Rebecchi MJ, Eberhardt R, Delaney T, Ali S, Bittman R
• Hydrolysis of short acyl chain inositol lipids by phospholipase C-delta 1.
• J Biol Chem. 1993; 268: 1735-41
• Display abstract

• We investigated the relationship between substrate aggregation and activation of phosphoinositide-specific phospholipase C-delta 1 (PLC-delta 1), isolated from bovine brain cytosol. The inositol lipids 1,2-dibutyryl-sn-glycero-3-phosphoinositol (di-C4-PI), 1,2-dihexanoyl-sn-glycero-3-phosphoinositol (di-C6-PI), and 1,2-dioctanoyl-sn-glycero-3-phosphoinositol (di-C8-PI) were prepared from synthetic cytidine diphosphate diglyceride analogs in a reaction with myo-inositol catalyzed by yeast phosphatidylinositol synthase. All three lipids served as substrates for PLC-delta 1 at concentrations significantly below their critical micelle concentration (cmc). Under these conditions, steps that might limit the reaction rate, such as membrane adsorption or penetration into the phospholipid surface, were eliminated. Below the cmc, the concentration of lipid substrate required to produce hydrolysis followed the order: di-C8-PI < di-C6-PI << di-C4-PI. Calcium was essential for hydrolysis of the short chain substrates at all lipid concentrations tested. The dependence of the reaction on calcium suggests that this ion activates PLC-delta 1 at a step other than adsorption to or penetration of the membrane surface. As the concentration of di-C8-PI was raised above the cmc, the reaction velocity increased 2-3-fold. These results are consistent with the idea that micellar or bilayer aggregates of phosphoinositol are not required for PLC-catalyzed hydrolysis, although the reaction rate is enhanced by micelle formation.

• Rhee SG, Lee CW, Jhon DY
• Phospholipase C isozymes and modulation by cAMP-dependent protein kinase.
• Adv Second Messenger Phosphoprotein Res. 1993; 28: 57-64

• Nieva JL, Goni FM, Alonso A
• Phospholipase C-promoted membrane fusion. Retroinhibition by the end-product diacylglycerol.
• Biochemistry. 1993; 32: 1054-8
• Display abstract

• The catalytic activity of phospholipase C induces fusion of pure lipid vesicles. When large unilamellar liposomes composed of phosphatidylcholine/phosphatidylethanolamine/cholesterol (2:1:1 mole ratio) are treated with phospholipase C, in the presence of 10 mM Ca2+, two enzyme effects can be distinguished: a fast one (half-time on the order of seconds) consisting mainly of vesicle-vesicle fusion and a slow one (half-time on the order of minutes) representing bulk lipid hydrolysis. The fast fusion process is inhibited by the end-product diacylglycerol, as well as by lysophosphatidylcholine and by low Ca2+ concentrations. The temperature dependence of enzyme activity (phospholipid hydrolysis), vesicle aggregation, and vesicle fusion (mixing of aqueous contents) has been separately studied. Enzyme activity and vesicle aggregation rates increase monotonically with temperature, while an optimum temperature is found for vesicle fusion, depending on liposome composition and assay conditions. The presence of diacylglycerol incorporated to the membrane (up to 10 mol %) does not produce any fusion effect even at temperatures as high as 80 degrees C: in situ diacylglycerol production by the enzyme appears to be required. The data are interpreted in support of a hypothesis according to which a "fusion intermediate" would be required, depending (among others) on bilayer composition, temperature, and Ca2+ concentration, for vesicle fusion to occur.

• Angelica Carrasco M, Sierralta J, Hidalgo C
• Phospholipase C activity in membranes and a soluble fraction isolated from frog skeletal muscle.
• Biochim Biophys Acta. 1993; 1152: 44-8
• Display abstract

• Highly purified triads and transverse tubules, as well as soluble fraction isolated from frog skeletal muscle, hydrolyze exogenous phosphatidylinositol 4,5-bisphosphate forming inositol 1,4,5-trisphosphate with maximal rates in the range 0.5-1 nmol/mg per min at pCa 3. Sarcoplasmic reticulum membranes present a minor activity. The hydrolysis rates in triads were 0.072 +/- 0.015 nmol/mg per min at pCa 7, increasing to 0.263 +/- 0.026 nmol/mg per min at pCa 5 with 1.0 mM Mg and 0.1 mM substrate. The phospholipase C activity of isolated transverse tubules at pCa 3 was 0.570 +/- 0.032 nmol/mg per min. Since triads contain 10% transverse tubules, and correcting for the small contribution of sarcoplasmic reticulum, the calculated phospholipase C activity of transverse tubules at pCa 3 is about 10-times higher than the observed values, suggesting loss of activity during isolation. The activation by calcium was also observed in a soluble fraction and was neither replaced nor inhibited by magnesium. No effect of GTP analogs on phospholipase C activity was detected.

• Ryan M, Huang JC, Griffith OH, Keana JF, Volwerk JJ
• A chemiluminescent substrate for the detection of phosphatidylinositol-specific phospholipase C.
• Anal Biochem. 1993; 214: 548-56
• Display abstract

• A water-soluble, chemiluminescent substrate for sensitive detection of bacterial phosphatidylinositol-specific phospholipase C (PI-PLC) activity was synthesized. This was achieved by replacing the diacylglycerol moiety found in the natural substrate, phosphatidylinositol, by a dioxetane-containing moiety, resulting in the synthetic substrate (+/-)-3-(4-methoxyspiro[1,2-dioxetane-3,2'- tricyclo[3.3.1.1(3,7]decan]-4-yl)phenyl myo-inositol-1-O-hydrogen phosphate (LUMI-PI). PI-PLC-catalyzed cleavage of the phosphodiester bond of LUMI-PI results in the formation of a chemiluminescent precursor, the labile anion AMP-D, which decays with emission of light. An assay procedure was developed using 96-well microtiter plates with detection of the chemiluminescence on autoradiography film. With this procedure the detection limit of purified phospholipase C was about 10 pg. Furthermore, PI-PLC activity was readily detected in situ using small quantities (< 1 microliters) of liquid cultures of PI-PLC-producing bacterial strains.

• Titball RW
• Bacterial phospholipases C.
• Microbiol Rev. 1993; 57: 347-66
• Display abstract

• A variety of pathogenic bacteria produce phospholipases C, and since the discovery in 1944 that a bacterial toxin (Clostridium perfringens alpha-toxin) possessed an enzymatic activity, there has been considerable interest in this class of proteins. Initial speculation that all phospholipases C would have lethal properties has not been substantiated. Most of the characterized enzymes fall into one of four groups of structurally related proteins: the zinc-metallophospholipases C, the sphingomyelinases, the phosphatidylinositol-hydrolyzing enzymes, and the pseudomonad phospholipases C. The zinc-metallophospholipases C have been most intensively studied, and lethal toxins within this group possess an additional domain. The toxic phospholipases C can interact with eukaryotic cell membranes and hydrolyze phosphatidylcholine and sphingomyelin, leading to cell lysis. However, measurement of the cytolytic potential or lethality of phospholipases C may not accurately indicate their roles in the pathogenesis of disease. Subcytolytic concentrations of phospholipase C can perturb host cells by activating the arachidonic acid cascade or protein kinase C. Nonlethal phospholipases C, such as the Listeria monocytogenes PLC-A, appear to enhance the release of the organism from the host cell phagosome. Since some phospholipases C play important roles in the pathogenesis of disease, they could form components of vaccines. A greater understanding of the modes of action and structure-function relationships of phospholipases C will facilitate the interpretation of studies in which these enzymes are used as membrane probes and will enhance the use of these proteins as models for eukaryotic phospholipases C.

• Ellis MV, Carne A, Katan M
• Structural requirements of phosphatidylinositol-specific phospholipase C delta 1 for enzyme activity.
• Eur J Biochem. 1993; 213: 339-47
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• Phosphatidylinositol-specific phospholipase C delta 1 isozyme of phosphoinositol-specific phospholipase C has been used for studies of structural requirements for the catalytic function. The enzyme was expressed in a bacterial system and purified to homogeneity. Using a combination of deletion mutant analysis and limited proteolysis, it was found that the large proportion of the molecule participated in formation of a catalytic domain (residues 139-756); it included regions of high and low conservation with other phospholipase-C molecules. These studies also showed that the residues spanning regions of conservation, designated as X and Y, were exposed and highly susceptible to proteolysis by trypsin. Two of the fragments resulting from the cleavage (30 kDa and 40 kDa) interacted and, under non-denaturing conditions, formed a protein of 70 kDa.

• James S, Demel R, Downes CP
• The activity of a phospholipase C beta assayed using phospholipid monolayers.
• Biochem Soc Trans. 1993; 21: 392-392

• Ueda H, Kobayashi T, Kishimoto M, Tsutsumi T, Okuyama H
• A possible pathway of phosphoinositide metabolism through EDTA-insensitive phospholipase A1 followed by lysophosphoinositide-specific phospholipase C in rat brain.
• J Neurochem. 1993; 61: 1874-81
• Display abstract

• Incubation of [2-3H]glycerol-labeled phosphatidylinositol with a crude cytosol fraction of rat brain in the presence of EDTA yielded [3H]lysophosphatidylinositol predominantly without accumulation of labeled monoacylglycerol and diacylglycerol. The pH optimum of this phospholipase A activity was 8.0. The activity for phosphatidylinositol was twofold higher than for phosphatidylethanolamine, whereas phosphatidylcholine, phosphatidylserine, and phosphatidic acid were not hydrolyzed significantly under the conditions used. The phospholipase A activity for phosphatidylethanolamine was resolved in part from that for phosphatidylinositol by ammonium sulfate fractionation of the cytosol, indicating the existence of at least two forms of EDTA-insensitive phospholipase A. The positional specificity of the phosphatidylinositol-hydrolyzing activity was found to be that of a phospholipase A1, as radioactive lysophosphatidylinositol was produced from 1-stearoyl-2-[1-14C]arachidonyl-sn-glycero-3-phosphoinositol without release of free arachidonate. A phospholipase C activity specific for lysophosphoinositides was found in a membrane fraction from rat brain, which was similar to that characterized in porcine platelets. The phospholipase C was demonstrated to hydrolyze the 2-acyl isomer as well as the 1-acyl isomer of lysophosphatidylinositol. Taken together, our results suggest a possible pathway through which phosphatidylinositol is selectively degraded to the 2-acyl isomer of lysophosphatidylinositol in a Ca(2+)-independent manner, and subsequently converted to a 2-monoacylglycerol in rat brain.

• Pawelczyk T, Lowenstein JM
• Inhibition of phospholipase C delta by hexadecylphosphorylcholine and lysophospholipids with antitumor activity.
• Biochem Pharmacol. 1993; 45: 493-7
• Display abstract

• The antineoplastic compound hexadecylphosphorylcholine (HPC) was shown to be a highly effective inhibitor of phospholipase C delta (PLC delta 1), with an I50 of about 30 nmol/mL (30 microM) in the presence and absence of 200 microM spermine. A number of lysophospholipids, of which HPC can be considered to be a structural analog, also inhibited PLC. Lysosphingomyelin, lysophosphatidylserine, and lysophosphatidylcholine exhibited I50 values of 15, 10, and 7 nmol/mL, respectively, in the presence of 200 microM spermine. The I50 values were increased to 21-53 nmol/mL in the absence of spermine. N,N-Dimethylsphingosine and N,N,N-trimethylsphingosine, which inhibit the metastatic potential of human and murine tumor cells, were weak activators of PLC delta 1. It is postulated that HPC is more effective as an antineoplastic agent than lysophospholipids because HPC is metabolized slowly, while the lysophospholipids are metabolized rapidly in vivo.

• Wakelam MJ et al.
• Phosphatidylcholine hydrolysis: a source of multiple lipid messenger molecules.
• Biochem Soc Trans. 1993; 21: 874-7

• Luk AS, Kaler EW, Lee SP
• Phospholipase C-induced aggregation and fusion of cholesterol-lecithin small unilamellar vesicles.
• Biochemistry. 1993; 32: 6965-73
• Display abstract

• We have investigated the effects of the Ca(2+)-requiring enzyme phospholipase C on the stability of sonicated vesicles made with different molar ratios of cholesterol to lecithin. Vesicle aggregation is detected by following turbidity with time. Upon the addition of phospholipase C and after a short lag period, the turbidity of a vesicle dispersion increases continuously with time. The rate of increase of turbidity increases with both the enzyme-to-vesicle ratio and the cholesterol content of the vesicles. Vesicle fusion and leakage of contents are monitored by a contents-mixing fusion assay using 8-aminonaphthalene-1,3,6-trisulfonic acid (ANTS) and p-xylylenebis(pyridinium bromide) (DPX) as the fluorescence probes [Ellens, H., Bentz, J. & Szoka, F.C. (1985) Biochemistry 24, 3099-3106]. The results clearly show that phospholipase C induces vesicle fusion. The rate of vesicle fusion correlates with the enzyme-to-vesicle ratio but not with the cholesterol content of the membrane. Negligible aggregation and fusion of vesicles occurs when the experiment is repeated with buffer free of Ca2+. The membrane-destabilizing diacylglycerol, a product of lecithin hydrolysis by phospholipase C, is speculated to play a major role in driving the observed vesicle aggregation and fusion. The kinetics of vesicle aggregation and vesicle fusion can be predicted by linking Michaelis-Menten enzyme kinetics to a mass-action model.

• Ivanov A, Kostadinova S
• Phosphatidylcholine-specific phospholipase C from Achromobacter xylosoxidans.
• Acta Microbiol Bulg. 1993; 29: 3-8
• Display abstract

• A new phosphatidylcholine-specific phospholipase C (EC 3.1.4.3.) has been isolated from the culture broth of Achromobacter xylosoxidans. Growth conditions were optimized for maximum production of the enzyme. A temperature-sensitive synthesis was established. Chromatography on CM Sephadex and polyacrylamide gel electrophoresis of the native enzyme revealed a number of isozymes. The water soluble substrate for phospholipase p-nitrophenylphosphorylcholine was not hydrolysed by the enzyme.

• Raymond FD, Moss DW, Fisher D
• Phase partitioning detects differences between phospholipase-released forms of alkaline phosphatase--a GPI-linked protein.
• Biochim Biophys Acta. 1993; 1156: 117-22
• Display abstract

• A number of enzymes are known to release alkaline phosphatase and other glycan phosphatidylinositol-anchored proteins from membrane surfaces. We describe a novel approach to detect and measure these activities by partitioning in aqueous phase systems. The procedures avoid the complications of micelle-formation involving hydrophobic molecules that may arise with detergent-based partition systems and can clearly distinguish between inositol-specific phospholipase C and D activities.

• Claro E, Fain JN, Picatoste F
• Noradrenaline stimulation unbalances the phosphoinositide cycle in rat cerebral cortical slices.
• J Neurochem. 1993; 60: 2078-86
• Display abstract

• Muscarinic cholinergic and alpha 1-adrenoceptor-mediated stimulation of phosphoinositide hydrolysis in rat cerebral cortex were compared by measuring carbachol- and noradrenaline-induced accumulation of various intermediates of the phosphoinositide cycle. Unlike carbachol, noradrenaline in the presence of guanosine 5'-O-(3-thiotriphosphate) did not stimulate phospholipase C activity in brain cortical membranes. In cortical slices, the efficacy of noradrenaline to stimulate accumulation of 3H-inositol phosphates and [32P]phosphatidic acid was 2.5 to threefold that of carbachol. However, noradrenaline was less effective than carbachol in stimulating accumulation of [3H]CDP-diacylglycerol and resynthesis of phosphatidylinositol. This was not due to calcium inhibition of CTP:phosphatidate cytidyltransferase or to different lithium requirements for carbachol- and noradrenaline-stimulated accumulation of [3H]CDP-diacylglycerol. The noradrenaline-induced unbalance of the phosphoinositide cycle, which was most apparent at relatively high concentrations of calcium (2.5 mM) in the incubation buffer, was qualitatively reproduced with ionomycin. The use of the alpha 1a-subtype-selective adrenoceptor antagonists WB4101 and 5-methylurapidil revealed a single alpha 1a-like component mediating the effects of noradrenaline. Our results suggest that the primary mechanism for phospholipase C activation by brain alpha 1 adrenoceptors involves an increase in intracellular calcium concentration.

• Rossi MA, Fidale F, Di Mauro C, Esterbauer H, Dianzani MU
• Effect of 4-hydroxy-2,3-trans-nonenal and related aldehydes on phospholipase C activity of rat neutrophils.
• Int J Tissue React. 1993; 15: 201-5
• Display abstract

• Our work has evaluated the effects of certain lipid peroxidation products, i.e. 4-hydroxy-2,3-trans-nonenal (HNE), 4-hydroxy-2,3-trans-octenal (HOE), 2-trans-nonenal and nonanal, on phosphoinositide-specific phospholipase C (PL-C). The enzymatic activity has been determined in vitro by measuring the hydrolysis of labelled phosphatidylinositol-4,5-bisphosphate added to plasma membranes isolated from rat neutrophils. Concentrations of HNE between 10(-8) and 10(-6) M, and concentrations of HOE ranging from 10(-11) to 10(-8) M, were able to activate PL-C. Neither 2-trans-nonenal nor nonanal induced any change of PL-C activity. HNE, HOE and 2-nonenal, but not nonanal, have been shown to possess chemotactic properties towards rat neutrophils. The good correlation between the concentrations of HNE and HOE active on PL-C and those able to stimulate cell migration suggests that their chemotactic activity might be mediated by the activation of PL-C, as in the case of most chemoattractants. On the contrary, this mechanism of action cannot be applied to 2-nonenal, an aldehyde much more hydrophobic than HNE or HOE; its chemotactic activity might be the consequence of some perturbation of the lipidic environment of the cell membrane where it dissolves easily.

• Hansen S, Hansen LK, Hough E
• The crystal structure of tris-inhibited phospholipase C from Bacillus cereus at 1.9 A resolution. The nature of the metal ion in site 2.
• J Mol Biol. 1993; 231: 870-6
• Display abstract

• We report here the crystal structure of the complex formed between phospholipase C (PLC) from Bacillus cereus and the widely used biochemical buffer tris (hydroxymethyl)-methylamine (Tris). The structure has been determined at 1.9 A resolution and refined to R = 20.3%. Tris has metal-binding properties, especially to Zn2+, and has been reported to reduce the activity of PLC. The amine nitrogen atom in Tris is co-ordinated to one of the three Zn2+ ions in the active site of the enzyme, thus confirming its chelating properties and the involvement of the metal ions in the catalytic process. The occupancy of the Zn2+ ion in site 2 in native PLC is 0.6 which could imply the presence of Ca2+ rather than Zn2+. The fact that Tris binds to this metal ion, the nature of the site 2 co-ordination shell and comparison with several homologous Zn-metalloenzymes indicate that PLC is a 3-Zn metalloenzyme. This study is one of a series which explores the active site of PLC by complexing the enzyme with inhibitors and substrate analogues.

• Flick JS, Thorner J
• Genetic and biochemical characterization of a phosphatidylinositol-specific phospholipase C in Saccharomyces cerevisiae.
• Mol Cell Biol. 1993; 13: 5861-76
• Display abstract

• Hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) by phosphatidylinositol-specific phospholipase C (PI-PLC) generates two second messengers, inositol 1,4,5-trisphosphate and 1,2-diacylglycerol. The polymerase chain reaction was used to isolate a Saccharomyces cerevisiae gene (PLC1) that encodes a protein of 869 amino acids (designated Plc1p) that bears greatest resemblance to the delta isoforms of mammalian PI-PLC in terms of overall sequence similarity and domain arrangement. Plc1p contains the conserved X and Y domains found in all higher eukaryotic PI-PLCs (51 and 29% identity, respectively, to the corresponding domains of rat delta 1 PI-PLC) and also contains a presumptive Ca(2+)-binding site (an E-F hand motif). Plc1p, modified by in-frame insertion of a His6 tract and a c-myc epitope near its amino terminus, was overexpressed from the GAL1 promoter, partially purified by nickel chelate affinity chromatography, and shown to be an active PLC enzyme in vitro with properties similar to those of its mammalian counterparts. Plc1p activity was strictly Ca2+ dependent: at a high Ca2+ concentration (0.1 mM), the enzyme hydrolyzed PIP2 at a faster rate than phosphatidylinositol, and at a low Ca2+ concentration (0.5 microM), it hydrolyzed PIP2 exclusively. Cells carrying either of two different deletion-insertion mutations (plc1 delta 1::HIS3 and plc1 delta 2::LEU2) were viable but displayed several distinctive phenotypes, including temperature-sensitive growth (inviable above 35 degrees C), osmotic sensitivity, and defects in the utilization of galactose, raffinose, and glycerol at permissive temperatures (23 to 30 degrees C). The findings reported here suggest that hydrolysis of PIP2 in S. cerevisiae is required for a number of nutritional and stress-related responses.

• Nozawa Y
• [Role of phosphatidylcholine breakdown by phospholipase C, D in signal transduction]
• Seikagaku. 1993; 65: 25-9

• Jones GA, Carpenter G
• The regulation of phospholipase C-gamma 1 by phosphatidic acid. Assessment of kinetic parameters.
• J Biol Chem. 1993; 268: 20845-50
• Display abstract

• A survey of lipids revealed that the anionic phospholipid phosphatidic acid activates both control and tyrosine-phosphorylated PLC-gamma 1. The mechanism by which phosphatidic acid activates both forms of PLC-gamma 1 was investigated using kinetic analysis. In the presence of phosphatidic acid, the substrate concentration response for control PLC-gamma 1 changes from sigmoidal to hyperbolic, while the cooperativity index decreases from 2.5 for control to 1.0 for tyrosine-phosphorylated PLC-gamma 1. The primary influence of phosphatidic acid on the control enzyme is on the cooperativity index and not the association of PLC-gamma 1 with substrate micelles, as phosphatidic acid had little effect on the micellar association constant, Ks. Phosphatidic acid also increases the activity of the tyrosine phosphorylated form of the enzyme. This increase is reflected in a decrease in the Km from 0.3- to 0.03-mol fraction phosphatidylinositol 4,5-bisphosphate. Phosphatidic acid has no effect on the Ks of the tyrosine-phosphorylated enzyme. From this data it is concluded that phosphatidic acid appears to activate PLC-gamma 1 by acting as an allosteric modifier.

• Kobayashi K, Shaver JK, Liang W, Siperstein AE, Duh QY, Clark OH
• Increased phospholipase C activity in neoplastic thyroid membrane.
• Thyroid. 1993; 3: 25-9
• Display abstract

• The phospholipase C (PLC)-protein kinase C (PKC) signal transduction pathway appears to be important for cellular growth of many normal and neoplastic tissues. Because alterations in the thyroid-stimulating hormone (TSH) receptor-adenylate cyclase-protein kinase A system in some thyroid tumors do not correlate with tumor size, invasiveness, or metastatic potential, we studied the PLC activity in both normal and neoplastic thyroid tissues from 11 patients. Five of these patients had follicular adenomas and 6 had papillary carcinomas. An 8,000 x g membrane fraction and a 105,000 x g cytosol fraction were prepared from the normal and neoplastic human thyroid tissues. PLC hydrolyzes phosphatidylinositol, 4,5-diphosphate (PIP2) to diacylglycerol (DAG) and inositol 1,4,5-triphosphate (IP3). Phospholipase C activity was determined measuring the hydrolysis of [3H]-PIP2. The activity of PLC in the neoplastic thyroid tissue membrane fraction (20.91 +/- 2.28 nmol PIP2 hydrolyzed/mg protein/120 min) was higher than that in normal thyroid membrane (14.27 +/- 0.82) (p < 0.05). In contrast, PLC activity was similar in the neoplastic (16.12 +/- 0.86 nmol PIP2 hydrolyzed/mg protein/120 min) and normal (16.66 +/- 0.60) cytosol. There was no difference between PLC activity in the membrane fraction from adenomas (21.21 +/- 3.71 nmol PIP2 hydrolyzed/mg protein/120 min) when compared with thyroid carcinomas (20.67 +/- 3.14). Neoplastic thyroid membranes have greater PLC activity than that found in normal thyroid membranes from the same patients. Although PLC activity in benign and malignant thyroid membranes was similar, the increased PLC activity in thyroid neoplasms may be responsible for or contribute to the enhanced growth of some thyroid tumors.

• Broomfield SJ, Hooper NM
• Characterization of an antibody to the cross-reacting determinant of the glycosyl-phosphatidylinositol anchor of human membrane dipeptidase.
• Biochim Biophys Acta. 1993; 1145: 212-8
• Display abstract

• A polyclonal antiserum raised to the phospholipase C-solubilized form of membrane dipeptidase (EC 3.4.13.11) purified from human kidney was found to cross-react with unrelated trypanosomal and porcine glycosyl-phosphatidylinositol anchored proteins. Those antibodies recognising the cross-reacting determinant (CRD) were isolated by chromatography on a column of immobilized phospholipase C-solubilized porcine aminopeptidase P (EC 3.4.11.9), and the epitopes involved in the recognition were then characterized by immunoelectrophoretic blot analysis and by a competitive ELISA. The phospholipase C-solubilized forms of human and porcine membrane dipeptidase, porcine aminopeptidase P and trypanosome variant surface glycoprotein were recognised by the anti-CRD antiserum, and this recognition was abolished by prior treatment of the proteins with either mild acid or nitrous acid. In contrast, the detergent-solubilized, membrane-forms of human and porcine membrane dipeptidase were not recognised. Of a range of components of the glycosyl-phosphatidylinositol anchor, only inositol 1,2-cyclic monophosphate and the insulin-mimetic disaccharide, glucosaminyl-1,6-inositol 1,2-cyclic monophosphate, inhibited in the micromolar range the binding of the anti-CRD antiserum to immobilized porcine aminopeptidase P. These results indicate that the major epitope recognised by this anti-CRD antiserum is the inositol 1,2-cyclic monophosphate formed on phospholipase C cleavage of the glycosyl-phosphatidylinositol anchor.

• Ogawara H, Higashi K, Manita S, Hidaka M, Kato H, Takenawa T
• An inhibitor of inositol-phospholipid-specific phospholipase C.
• Biochim Biophys Acta. 1993; 1175: 289-92
• Display abstract

• Q12713 substance, a cyclic peptide from Actinomadura spp., strongly inhibited the enzyme activity of phosphatidylinositol-4,5-bisphosphate-specific phospholipase C (PIP2-PLC) but scarcely inhibited other phospholipases. Kinetic analysis demonstrated that the inhibition of PIP2-PLC activity was competitive with respect to phosphatidylinositol 4,5-bisphosphate. Calcium and magnesium ions had no significant effect on the inhibitory activity. On the contrary, potassium or rubidium ion was essential for the inhibitory activity. Furthermore, NaF and AlCl3-stimulated increase of phosphoinositides was decreased by Q12713 in the cultured 3T3 cells.

• Brophy CM, Mills I, Rosales O, Isales C, Sumpio BE
• Phospholipase C: a putative mechanotransducer for endothelial cell response to acute hemodynamic changes.
• Biochem Biophys Res Commun. 1993; 190: 576-81
• Display abstract

• Endothelial cells (EC) in vivo are exposed to a multitude of physical forces with each pulse of the cardiac cycle. Ongoing studies support the concept that EC respond to these forces through specific signal transduction pathways. Previous investigations in our laboratory have shown that EC respond to the initiation of cyclic strain or to an acute increase in cyclic strain frequency with the production of inositol 1,4,5-trisphosphate (IP3). This study demonstrates that EC also respond to an acute decrease in cyclic stretch frequency with a transient increase in IP3 production. Thus, EC detect both increases and decreases in cyclic stretch frequency with phospholipase C (PLC) activation leading to IP3 generation.

• Simoes AP, Schnabel P, Pipkorn R, Camps M, Gierschik P
• A peptide corresponding to a potential polyphosphoinositide binding site of phospholipase C-beta 2 enhances its catalytic activity.
• FEBS Lett. 1993; 331: 248-51
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• A peptide corresponding to a basic consensus amino acid motif present in both actin-binding proteins and phosphoinositide-specific phospholipases C was synthesized and its effect on the activity of a recombinant phospholipase C-beta 2 (PLC beta 2) expressed in baculovirus-infected insect cells was studied. The peptide markedly and specifically stimulated the activity of the enzyme. This stimulatory effect required a particular primary and/or secondary structure of the peptide and occurred without lowering the affinity of the enzyme for Ca2+. The function of the PLC beta 2 segment corresponding to the peptide might be to bind and offer the substrate to the catalytic domain of this enzyme in a more favorable configuration or, alternatively, to interact with a hypothetical inhibitory constraint.

• Challiss RA, Wilkes LC, Patel V, Purkiss JR, Boarder MR
• Phospholipase D activation regulates endothelin-1 stimulation of phosphoinositide-specific phospholipase C in SK-N-MC cells.
• FEBS Lett. 1993; 327: 157-60
• Display abstract

• Endothelin-1 (ET-1) is known to stimulate phospholipase C (PLC) activity in SK-N-MC human neuroblastoma/epithelioma cells: here we show that phospholipase D (PLD) is also stimulated. The generation of inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) by ET-1-stimulated PLC was attenuated by protein kinase C (PKC) activation and enhanced by PKC inhibition. An enhancement of ET-1-stimulated Ins(1,4,5)P3 accumulation was also seen when the product of PLD activity was either diverted into phosphatidyl butanol in the presence of butanol, or phosphatidate phosphohydrolase (PPH) activity was inhibited by DL-propranolol. We conclude that there is an inhibitory, PKC-mediated, feedback loop in these cells which is dependent, in part, on the activation of PKC by product(s) of the PLD/PPH pathway. This provides a novel role for agonist-stimulated PLD activation.

• Lewis KA, Garigapati VR, Zhou C, Roberts MF
• Substrate requirements of bacterial phosphatidylinositol-specific phospholipase C.
• Biochemistry. 1993; 32: 8836-41
• Display abstract

• A series of symmetric short-chain phosphatidylinositols (PI), including dihexanoyl-PI, diheptanoyl-PI (racemic as well as D and L forms), and 2-methoxy inositol-substituted diheptanoyl-PI, have been synthesized, characterized, and used to investigate key mechanistic questions about phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus thuringiensis. Key results include the following: (i) bacterial PI-PLC exhibits a 5-6-fold "interfacial activation" when its substrate is present in an interface as opposed to existing as a monomer in solution (in fact, the similarity to the activation observed with nonspecific PLC enzymes suggests a similarity in activation mechanisms); (ii) the 2-OH must be free since the enzyme cannot hydrolyze diheptanoyl-2-O-methyl-PI (this is most consistent with the formation of inositol cyclic 1,2-phosphate as a necessary step in catalysis); (iii) the inositol ring must have the D stereochemistry (the L-inositol attached to the lipid moiety is neither a substrate nor an inhibitor); and (iv) the presence of noninhibitory L-PI with the D-PI substrate relieves the diacylglycerol product inhibition detected at approximately 30% hydrolysis.

• Ferreira PA, Shortridge RD, Pak WL
• Distinctive subtypes of bovine phospholipase C that have preferential expression in the retina and high homology to the norpA gene product of Drosophila.
• Proc Natl Acad Sci U S A. 1993; 90: 6042-6
• Display abstract

• The Drosophila norpA gene encodes a phospholipase C involved in phototransduction. However, phospholipase C apparently is not directly involved in phototransduction in vertebrate photoreceptors, although light-activated phospholipase C activity has been reported in vertebrate rod outer segments. Conserved regions of norpA cDNA were used to isolate bovine cDNAs that would encode four alternative forms of phospholipase C of the beta class that are highly homologous to the norpA protein and expressed preferentially in the retina. Two of the variants are highly unusual in that they lack much of the N-terminal region present in all other known phospholipases C. The sequence conservation between these proteins and the norpA protein is higher than that between any other known phospholipases C. GTPase sequence motifs found in proteins of the GTPase superfamily are found conserved in all four variants of the bovine retinal protein as well as the norpA protein but not in other phospholipases C. Results suggest that these proteins together with the norpA protein constitute a distinctive subfamily of phospholipases C that are closely related in structure, function, and tissue distribution. Mutations in the norpA gene, in addition to blocking phototransduction, cause light-dependent degeneration of photoreceptors. In view of the strong similarity in structure and tissue distribution, a defect in these proteins may have similar consequences in the mammalian retina.

• Calderwood SK, Stevenson MA
• Inducers of the heat shock response stimulate phospholipase C and phospholipase A2 activity in mammalian cells.
• J Cell Physiol. 1993; 155: 248-56
• Display abstract

• Although the mammalian heat shock response has been well characterized, the processes that mediate the induction of the response and the regulation of heat shock protein function are not completely understood. We have investigated the potential role in heat-shocked cells of phosphoinositide-specific phospholipase C (PLC), a membrane enzyme activity involved in transmembrane signal transduction. Our studies indicate that heat shock activates PLC in each of seven cell lines, including cells of human, rat, mouse, and hamster origin. Heat shock produced increases in inositol phosphate concentrations comparable in magnitude to those achieved after simulation with growth factors, indicating that heat shock might initiate transmembrane signaling cascades of potential importance in cellular regulation. Common cellular responses to heat and growth factors also included feedback modulation of PLC by its products and the parallel stimulation of phospholipase A2 activity. In addition to heat shock, other agents that induce the stress response stimulated PLC activity. The data indicate a close correlation between expression of the mammalian heat shock response and stimulation of PLC activity and indicate a possible role for this enzyme activity in the regulation of some aspects of the stress response.

• Kamberov ES, Trifonov B
• Altered levels of phosphatidylinositol phospholipase C activity in rat liver cells in response to insulin, epinephrine, acetylcholine and bacterial phospholipase C.
• Regul Pept. 1993; 44: 257-67
• Display abstract

• Rat liver cells were homogenized and subsequently fractionated by a simplified method based on microfiltration, which proved to give a high recovery of membrane-bound phosphatidylinositol phospholipase C (PI-PLC) activity. The effects of insulin, acetylcholine (AC), epinephrine (EN) and bacterial phospholipase C (bPLC) on the PI-PLC activity were studied after in vitro treatment of isolated membranes or after in situ application in rat liver. A dose-dependent increase of membranous PI-PLC (up to 3-fold) and corresponding 36 to 72% decline of the cytosolic activity were established upon treatment with supraphysiological doses of insulin or with bPLC, respectively. AC induced a biphasic response with a maximal stimulation in the micromolar range. On the other hand EN promoted a slight but significant dose-dependent inhibition of PI-PLC in both cytosol and membranes. Sodium fluoride was also a potent inhibitor of the membrane-associated PI-PLC with an EC50 value of about 5 mM. The combined assay with NaF and EN revealed no additivity between their inhibitory effects, suggesting a common step in the mechanism(s) of inhibition caused by the two agents. The stimulatory effects of insulin and AC were partially reduced by soluble cytosolic factors, which still remain to be identified. When insulin and AC were applied in combination in the presence of cytosol, this resulted in a 56% inhibition of PI-PLC below the control level.

• Kuroki M, Kuwahara M, Tsuruta Y, Murakami M, Matsuoka Y
• Effective purification of nonspecific cross-reacting antigens with phosphatidylinositol-specific phospholipase C.
• Prep Biochem. 1993; 23: 333-49
• Display abstract

• Two molecular species of nonspecific cross-reacting antigens, NCA-90 and NCA-50 with mol. wts. of 90,000 and 50,000, respectively, were effectively extracted with phosphatidylinositol-specific phospholipase C (PI-PLC) from human lung tissues, followed by extraction with perchloric acid, immunoaffinity chromatography with anti-NCA adsorbent, and gel filtration on a TSK G3000SW column. The yields of NCA were about 2 times more than those obtained by the usual method without PI-PLC. Addition of 0.05 unit of PI-PLC to 1 g of lung tissue and incubation at 37 degrees C for 1 h with continuous shaking seem to be practically sufficient for NCA extraction. The immunochemical properties of the NCAs thus obtained were found to be identical to those of NCAs obtained by the ordinary method.

• Bullock TL, Ryan M, Kim SL, Remington SJ, Griffith OH
• Crystallization of phosphatidylinositol-specific phospholipase C from Bacillus cereus.
• Biophys J. 1993; 64: 784-91
• Display abstract

• Phosphatidylinositol-specific phospholipase C (PI-PLC) cleaves phosphoinositides into two parts, lipid-soluble diacylglycerol and the water-soluble phosphorylated inositol. Two crystal forms of Bacillus cereus PI-PLC have been obtained by the vapor diffusion technique. Hexagonal crystals were grown from solutions containing polyethylene glycol (PEG; 4,000 to 8,000 D). The space group of these hexagonal crystals is P6(1)22 (or the enantiomorphic space group P6(5)22), with cell constants a = b = 133 A, and c = 231 A. The crystals diffract to 2.8 A. The second crystalline form was grown from a two-phase PEG (600 D)-sodium citrate solution. The phase diagram and PI-PLC distribution between phases has been determined. The enzyme crystallizes from the PEG-rich phase. The crystals are orthorhombic with space group P2(1)2(1)2(1) (a = 45 A, b = 46 A, c = 160 A), and contain one PI-PLC monomer per asymmetric unit. The orthorhombic crystals diffract to 2.5 A. Both the hexagonal and orthorhombic forms are suitable for crystallographic studies.

• Hansen S, Hough E, Svensson LA, Wong YL, Martin SF
• Crystal structure of phospholipase C from Bacillus cereus complexed with a substrate analog.
• J Mol Biol. 1993; 234: 179-87
• Display abstract

• We report the first crystal structure of a complex between PLC from Bacillus cereus (PLCBc) and a competitive inhibitor that is an analog of the natural phospholipid substrate. The structure has been determined at 1.9 A resolution and refined to a final R-factor of 15.7%. The inhibitor binds with its phosphonyl group to the three Zn ions in the active site of the enzyme and is also involved in a hydrogen bonded network including several water molecules and amino acid side-chains which appear to help orient the substrate for productive binding. The interactions within this complex provide some important information regarding the mechanism of PLC-catalyzed hydrolysis of membrane phospholipids. A water molecule, located approximately apical to the diacylglycerol leaving group, seems to be the most likely candidate for the attacking nucleophile which initiates the reaction.

• Volwerk JJ, Birrell GB, Hedberg KK, Griffith OH
• A high level of cell surface phosphatidylinositol-specific phospholipase C activity is characteristic of growth-arrested 3T3 fibroblasts but not of transformed variants.
• J Cell Physiol. 1992; 151: 613-22
• Display abstract

• Confluent monolayers of four contact-inhibited mouse fibroblast lines (Swiss 3T3, Balb/c 3T3, NIH 3T3, and C3H10T1/2) were found to have substantial levels of a cell surface phosphatidylinositol-specific phospholipase C (ecto-PLC). In contrast, confluent cultures of virally, chemically, or spontaneously transformed variants derived from these cell lines expressed undetectable or negligible levels of this enzyme activity. A simple and rapid assay, using lysophosphatidylinositol radio-labeled in the inositol group ([3H]-lysoPI) as the substrate was developed to provide a quantitative measure of the phospholipase C activity present at the external cell surface. For cells testing positive for ecto-PLC activity, rapid uptake of [3H]-lysoPI is accompanied by the simultaneous appearance of [3H]-inositol phosphate in the external medium. Confluent monolayers of the four mouse fibroblast lines exhibiting density-dependent growth inhibition had levels of ecto-PLC activity in the range of 50-800 pmol/min/10(6) cells (i.e., about 20-50 times greater than the activity observed for the transformed variants). The expression of ecto-PLC activity at the cell surface of the Swiss or Balb/c cells was dependent on the state of cell proliferation. Cultures which had become quiescent through attainment of confluence displayed a tenfold increased activity over that of subconfluent, growing cultures of these cells. Similarly, subconfluent Swiss 3T3 cells which had become quiescent following exposure to low serum conditions also showed increased activity. These results indicate that there may exist a correlation between the control of cell proliferation in contact-inhibited mouse fibroblasts and the expression of inositol phospholipid-specific phospholipase C activity at the external cell surface.

• Kuriki H, Tamiya-Koizumi K, Asano M, Yoshida S, Kojima K, Nimura Y
• Existence of phosphoinositide-specific phospholipase C in rat liver nuclei and its change during liver regeneration.
• J Biochem (Tokyo). 1992; 111: 283-6
• Display abstract

• We found phosphoinositide-specific phospholipase C (PtdIns-PLC) activity in nuclei isolated from rat liver. The enzyme hydrolyzed phosphatidylinositol, phosphatidylinositol 4-monophosphate (PIP) and phosphatidylinositol 4,5-bisphosphate in a Ca(2+)-dependent manner, and produced inositol mono-, bis-, and triphosphate, respectively. Neither phosphatidylcholine, phosphatidylethanolamine, nor phosphatidylserine was utilized as a substrate. After partial hepatectomy, the PtdIns-PLC activity in isolated nuclei increased transiently in the S phase (20-22 h post-hepatectomy), to 2.5-fold higher than in the control, when measured with PIP. This result suggests a close relationship between the nuclear PtdIns-PLC, especially its PIP-hydrolyzing activity, and cell proliferation.

• Hansen S, Hansen LK, Hough E
• Crystal structures of phosphate, iodide and iodate-inhibited phospholipase C from Bacillus cereus and structural investigations of the binding of reaction products and a substrate analogue.
• J Mol Biol. 1992; 225: 543-9
• Display abstract

• The crystal structure of the complex formed between phospholipase C (PLC) from Bacillus cereus and inorganic phosphate (Pi), which is an inhibitor, has been determined and refined to 2.1 A resolution. The final R-factor is 19.7%. We have also studied the binding of two other inhibitors, iodide and iodate, to PLC. X-ray data for these two complexes were collected to 2.8 A resolution during the search for heavy-atom derivatives. A series of screening experiments where PLC crystals have been treated with several reaction products and a substrate analogue were carried out to clarify the question of substrate binding. The results have so far been ambiguous but are discussed briefly. Phosphate and iodate are both found to bind to the three metal ions in the protein molecule, suggesting that these ions are involved directly in the catalytic process and thereby identifying the active site. PLC also binds nine iodide ions, eight of which are on the surface of the molecule and of lower occupancy. The ninth blocks the entrance to the active site cleft and is of higher occupancy. Altogether, these results suggest that the substrate, a phospholipid, is associated directly with the metal ions during catalysis.

• Jones G, Carpenter G
• Regulation of phospholipase C isozymes.
• Prog Growth Factor Res. 1992; 4: 97-106
• Display abstract

• Phosphatidylinositol bisphosphate hydrolysis is an immediate response to many hormones, including growth factors. The hydrolysis of phosphatidylinositol bisphosphate is catalyzed by phosphatidylinositol-specific phospholipase C. A number of phospholipase C isozymes have been identified. Different isozymes are activated by different receptor classes. This review will summarize the different isozymes of phospholipase C, and the current knowledge of the mechanisms by which phospholipase C activity is modulated by growth factors.

• Rhee SG, Choi KD
• Regulation of inositol phospholipid-specific phospholipase C isozymes.
• J Biol Chem. 1992; 267: 12393-6

• Rebecchi M, Boguslavsky V, Boguslavsky L, McLaughlin S
• Phosphoinositide-specific phospholipase C-delta 1: effect of monolayer surface pressure and electrostatic surface potentials on activity.
• Biochemistry. 1992; 31: 12748-53
• Display abstract

• We added phospholipase C-delta 1 (PLC-delta) to the aqueous subphase beneath monolayers formed from mixtures of phosphatidylinositol 4,5-bisphosphate (2% PIP2), phosphatidylserine (33% PS), and phosphatidylcholine (65% PC) and then measured the initial rate of hydrolysis of PIP2 after addition of 10 microM free calcium. Increasing the surface pressure of the monolayer, pi, from 20 to 40 mN/m decreased the rate of hydrolysis 200-fold. The rate of hydrolysis depends exponentially on the surface pressure: rate alpha exp(-pi Ap/kT) where k is the Boltzmann constant, T is the temperature, and Ap congruent to 1 nm2. Similar results were obtained with different (1 and 100 microM) free [Ca2+] and with different mole fractions of PIP2. The results are consistent with a model in which PLC-delta binds to PIP2 with high affinity (Ka = 10(6) M-1) in the absence of calcium ions [Rebecchi, M.J., Peterson, A., & McLaughlin, S. (1993) Biochemistry (preceding paper in this issue)], and a portion of PLC-delta of area Ap inserts into the monolayer doing work = pi Ap prior to hydrolysis of PIP2. Removing the monovalent acidic lipid PS from the monolayer decreases the activity of PLC-delta 4-fold, this effect of PS on activity is similar to the effect of monovalent acidic lipids on the binding of PLC-delta to PIP2 in bilayer vesicles.

• Byberg JR, Jorgensen FS, Hansen S, Hough E
• Substrate-enzyme interactions and catalytic mechanism in phospholipase C: a molecular modeling study using the GRID program.
• Proteins. 1992; 12: 331-8
• Display abstract

• Based on the high-resolution X-ray crystallographic structure of phospholipase C from Bacillus cereus, the orientation of the phosphatidylcholine substrate in the active site of the enzyme is proposed. The proposal is based on extensive calculations using the GRID program and molecular mechanics geometry relaxations. The substrate model has been constructed by successively placing phosphate, choline and diacylglycerol moieties in the positions indicated from GRID calculations. On the basis of the resulting orientation of a complete phosphatidylcholine molecule, we propose a mechanism for the hydrolysis of the substrate.

• Wakui H et al.
• Localization of phosphoinositide-specific phospholipase C-alpha in porcine kidney.
• Kidney Int. 1992; 42: 888-95
• Display abstract

• The presence of multiple forms of phosphoinositide-specific phospholipase C (PLC), an important enzyme in the cell signal transduction, suggests that specialized functions of tissues and cells may require different modes of PLC regulation. In the present study, we have purified a 54-kDa heparin-binding protein from a 4 M guanidine hydrochloride extract of porcine kidney, and identified it as one of isoenzymes of PLC on the basis of its partial amino acid sequence. Among 194 determined sequences of the porcine protein, 186 residues were identical with those deduced from nucleotide sequence of the cDNA encoding rat PLC-alpha. The subcellular distribution of porcine renal PLC-alpha was examined by Western blotting by using a specific antibody against the purified protein. Quantitation of the Western blots revealed that 70% of PLC-alpha was membrane-associated. Immunohistochemical studies showed a specific localization of PLC-alpha in epithelial cells of distal tubules and collecting ducts of normal porcine kidney, but not in other cells composing the nephron. Moreover, the highest expression of PLC-alpha was observed in apical membranes in these epithelial cells. Thus, this form of PLC is considered to have a specific role in the signal transduction process related to regional renal tubular functions.

• Shashidhar MS, Volwerk JJ, Griffith OH, Keana JF
• A chromogenic substrate for phosphatidylinositol-specific phospholipase C: 4-nitrophenyl myo-inositol-1-phosphate.
• Chem Phys Lipids. 1991; 60: 101-10
• Display abstract

• A chromogenic water-soluble substrate for phosphatidylinositol-specific phospholipase C was synthesized starting from myo-inositol employing isopropylidene and 4-methoxytetrahydropyranyl protecting groups. In this analogue of phosphatidylinositol, 4-nitrophenol replaces the diacylglycerol moiety, resulting in synthetic, racemic 4-nitrophenyl myo-inositol-1-phosphate. Using this synthetic substrate a rapid, convenient and sensitive spectrophotometric assay for the phosphatidylinositol-specific phospholipase C from Bacillus cereus was developed. Initial rates of the cleavage of the nitrophenol substrate were linear with time and the amount of enzyme used. At pH 7.0, specific activities for the B. cereus enzyme were 77 and 150 mumol substrate cleaved min-1 (mg protein)-1 at substrate concentrations of 1 and 2 mM, respectively. Under these conditions, less than 50 ng quantities of enzyme were easily detected. The chromogenic substrate was stable during long term storage (6 months) as a solid at -20 degrees C.

• Yamada M, Mizuguchi M, Rhee SG, Kim SU
• Developmental changes of three phosphoinositide-specific phospholipase C isozymes in the rat nervous system.
• Brain Res Dev Brain Res. 1991; 59: 7-16
• Display abstract

• Developmental changes of 3 phosphoinositide-specific phospholipase C isozymes (PI-PLC-beta, PI-PLC-gamma and PI-PLC-delta) in the rat nervous system were studied by immunohistochemical and immunochemical methods. PI-PLC-gamma immunoreactivity was intensely expressed in the radial fibers from the late fetal to early newborn stages, while weaker PI-PLC-beta reaction was also demonstrated in these structures. PI-PLC-beta and PI-PLC-gamma immunoreactivity appeared in neurons of various regions after the first postnatal week and then increased to the adult stage. Bergmann glia and some astrocytes also showed weak immunoreactivity for both isozymes from the newborn stage, while such immunoreactive astrocytes were relatively restricted in distribution in the white matter and hippocampus at the adult stage. PI-PLC-delta immunoreactivity appeared in astrocytes of entire cerebral regions from the second postnatal week, although weak antigenicity was also present in some neurons. Immunoblot analysis revealed that the immunoreactivities of 3 PI-PLC isozymes were present in both fetal and adult brains, with strong reactions of PI-PLC-beta and PI-PLC-delta in adult brain and that of PI-PLC-gamma in fetal brain. These results suggest that each PI-PLC isozyme plays important roles in different cell types in the course of their differentiation, and that some PI-PLC isozymes, especially PI-PLC-gamma, may be involved in cellular division and growth during the histogenesis of the central nervous system.

• Haber MT, Fukui T, Lebowitz MS, Lowenstein JM
• Activation of phosphoinositide-specific phospholipase C delta from rat liver by polyamines and basic proteins.
• Arch Biochem Biophys. 1991; 288: 243-9
• Display abstract

• Phospholipase C from rat liver with a molecular weight of 87,000 (PLC delta) is stimulated by polyamines, basic proteins, and basic polyamino acids. The activation occurs in both the presence and the absence of detergents. Half-maximum activation by spermine is observed at 0.15 mM, with optimum effects between 0.2 and 0.5 mM. Spermine inhibits above 0.5 mM. Half-maximum activation by spermidine and putrescine is observed at 0.9 and 6 mM, respectively, with optimum effects at 2 and 5 mM, respectively. These polyamines also inhibit at higher concentrations. Neomycin activates the enzyme with an optimum concentration of 10 microM, but maximum activation is less than with polyamines. Half-maximum activation by histone 2B occurs at 0.5 micrograms/ml (36 nM), with maximum stimulation at 1.5 micrograms/ml. Other histones, protamine, melittin, poly-L-ornithine, poly-L-lysine, poly-D-lysine, and poly-L-arginine, activate optimally at 3-10 micrograms/ml. Myelin basic protein and lysozyme activate optimally at 50-100 micrograms/ml. Typical activations are three- to eightfold, but under some conditions the enzyme shows little or no activity in the absence of basic activators. The basic activators lower the salt concentration required for maximal activity. In the case of the detergent-micelle assay, histone shifts the optimum NaCl concentration from 350 to 200 mM for PIP2, from 260 to 100 mM for PIP, and from 150 to 0 mM for PI. Histone potentiates the activation by Ca2+, but does not shift the optimum Ca2+ concentration. The optimum salt and Ca2+ concentrations are linked, such that a decrease in the concentration of one decreases the optimum concentration of the other. Activation by histone is diminished by MgCl2 in a concentration-dependent manner.

• Okano Y, Banno Y, Matsui A
• [Phospholipase C: its structure and molecular diversity]
• Tanpakushitsu Kakusan Koso. 1991; 36: 270-9

• Shashidhar MS, Volwerk JJ, Keana JF, Griffith OH
• A fluorescent substrate for the continuous assay of phosphatidylinositol-specific phospholipase C: synthesis and application of 2-naphthyl myo-inositol-1-phosphate.
• Anal Biochem. 1991; 198: 10-4
• Display abstract

• A fluorescent water-soluble substrate for phosphatidylinositol-specific phospholipase C was synthesized. The diacylglycerol moiety of the natural substrate, phosphatidylinositol, was replaced by the fluorescent moiety, 2-naphthol, resulting in the synthetic substrate, racemic 2-naphthyl myo-inositol-1-phosphate. The synthetic substrate provided a continuous fluorometric assay for the phosphatidylinositol-specific phospholipase C from Bacillus cereus. Initial rates of the cleavage of the 2-naphthyl substrate by the phospholipase measured by fluorometry were linear with time and the amount of enzyme added. The specific enzyme activity at pH 8.5 and 25 degrees C was about 0.04 mumol/min mg protein at an initial substrate concentration of 0.8 mM. 31P NMR experiments suggest that, as with phosphatidylinositol itself, cleavage of the fluorescent substrate proceeds in two steps via a myo-inositol-1,2-cyclic phosphate intermediate, and that only the D-isomer is a substrate for the B. cereus phospholipase. The synthetic substrate was stable during long-term storage as a solid in the dark at -20 degrees C. It was also stable for several weeks when stored in the dark frozen in aqueous solution near neutral pH.

• Tompkins TA, Moscarello MA
• A 57-kDa phosphatidylinositol-specific phospholipase C from bovine brain.
• J Biol Chem. 1991; 266: 4228-36
• Display abstract

• A phosphatidylinositol-specific phospholipase C (PI-PLC) has been isolated from bovine brain (purification factor of 5.6 x 10(4)). By sodium dodecyl sulfate-polyacrylamide gel electrophoresis, it had a Mr of 57,000. Neither amino nor neutral sugars were detected in the purified enzyme. The pH optimum was 7.0-7.5, and the activity decreased only slightly at pH 8.0. When phosphatidylinositol was used as a substrate, the optimum Ca2+ requirement was 4 mM, and Km was 260 microM. When phosphatidylinositol 4,5-bisphosphate was used, the optimum Ca2+ requirement was 10(-7) M, and the Km was reduced to 90 microM. Lipid specificity studies showed that equal amounts of inositol phosphate and diacylglycerol were released from phosphatidylinositol but 4 times as much inositol 1,4,5-trisphosphate was released from phosphatidylinositol 4,5-bisphosphate. Other lipids, phosphatidylcholine, phosphatidylethanolamine, and sphingomyelin, were not substrates. Failure to detect phosphatidic acid confirmed the absence of a phospholipase D activity in the purified enzyme. Myelin basic protein (MBP) stimulated the PI-PLC activity between 2- and 3-fold. Histone had a small effect only, whereas bovine serum albumin and cytochrome C had no effect. Phosphorylation of MBP reduced the stimulatory effect. Protein-protein interactions between MBP and PI-PLC have been demonstrated both immunologically and by sucrose density gradients. A stoichiometry of 1:1 has been suggested by the latter method. A number of peptides have been prepared by chemical, enzymatic, and synthetic methods. Peptides containing the MBP sequences consisting of residues 24-33 and 114-122 stimulated the PI-PLC but were less effective than the intact protein.

• Smith MR, Liu YL, Kim H, Rhee SG, Kung HF
• Inhibition of serum- and ras-stimulated DNA synthesis by antibodies to phospholipase C.
• Science. 1990; 247: 1074-7
• Display abstract

• Several immunologically distinct isozymes of inositol phospholipid-specific phospholipase C (PLC) have been purified from bovine brain. Murine NIH 3T3 fibroblasts were found to express PLC-gamma, but the expression of PLC-beta was barely detectable by radioimmunoassay or protein immunoblot. A mixture of monoclonal antibodies was identified that neutralizes the biological activity of both endogenous and injected purified PLC-gamma. When co-injected with oncogenic Ras protein or PLC-gamma, this mixture of antibodies inhibited the induction of DNA synthesis that characteristically results from the injection of these proteins into quiescent 3T3 cells. However, when oncogenic Ras protein or PLC-gamma was co-injected with a neutralizing monoclonal antibody to Ras, only the DNA synthesis induced by the Ras protein was inhibited--that induced by PLC was unaffected. These results suggest that the Ras protein is an upstream effector of PLC activity in phosphoinositide-specific signal transduction and that PLC-gamma activity is necessary for Ras-mediated induction of DNA synthesis.

• Shashidhar MS, Volwerk JJ, Keana JF, Griffith OH
• Inhibition of phosphatidylinositol-specific phospholipase C by phosphonate substrate analogues.
• Biochim Biophys Acta. 1990; 1042: 410-2
• Display abstract

• Non-hydrolysable analogues of phosphatidylinositol were synthesized and tested as inhibitors of phosphatidylinositol-specific phospholipase C from Bacillus cereus. In these molecules, the phosphodiester bond of phosphatidylinositol hydrolyzed by the phospholipase was replaced by a phosphonate linkage and a simpler hydrophobic group replaced the diacylglycerol moiety. One of the phosphonates also contained a carboxylate functional group suitable for matrix attachment. All three synthetic phosphonates inhibited the phospholipase C activity in a concentration-dependent manner, with the analogue most closely resembling the structure of the natural substrate, phosphatidylinositol, being the most potent inhibitor. The data indicate that phosphonate analogues of phosphatidylinositol may be useful for study of phospholipase C and other proteins interacting with myo-inositol phospholipids.

• Langeland N, Moore LJ
• Reduction of HSV-1 binding to BHK cells after treatment with phosphatidylinositol-specific phospholipase C.
• FEBS Lett. 1990; 277: 253-6
• Display abstract

• Baby-hamster kidney cells were treated with unspecific and phosphatidylinositol-specific phospholipase C (PI-PLC) prior to infection with herpes simplex virus type 1 or 2. Subsequent to PI-PLC treatment, a 30% reduction of infectivity and receptor binding was observed for type 1 virus, while type 2 was unaffected. Treating the cells with unspecific phospholipase C did not affect subsequent infection with either virus. Treatment of the virus particles with the unspecific phospholipase reduced its infectivity, probably due to loss of the viral envelope. PI-PLC treatment of virus particles did not have any such effect on virus infectivity.

• Fain JN
• Regulation of phosphoinositide-specific phospholipase C.
• Biochim Biophys Acta. 1990; 1053: 81-8
• Display abstract

• The receptors involved in the regulation of phospholipase C by hormones, neurotransmitters and other ligands have seven transmembrane-spanning hydrophobic regions (seven-helix motif) and no known enzymatic activity. Furthermore these receptors can be isolated as complexes with guanine nucleotide binding (G) proteins. Guanine nucleotides affect the binding of hormones that stimulate phospholipase C and it has been possible to see activation of GTPase activity in membranes upon addition of these ligands. Further indirect evidence for a Gp (p stands for phospholipase C activation) protein is the finding that in membranes agonist activation of phospholipase C requires the presence of GTP gamma S a non-hydrolyzable analog of GTP. Furthermore, fluoride is able to activate phospholipase C but its inhibition of phosphatidylinositol-4' kinase (PI-4' kinase) can interfere with efforts to demonstrate this in intact cells. There are four major isozymes of phospholipase C that have been cloned and sequenced. Recently it was found that phospholipase C-gamma as well as PI-3'-kinase are substrates for phosphorylation on tyrosine residues by the EGF and PDGF receptors. The PI-3' kinase is able to convert phosphatidylinositol 4,5-bisphosphate (PIP2) to phosphatidylinositol 3,4,5-trisphosphate (PIP3) but the function of this lipid is unknown since it is not a substrate for any known phospholipase C. While much has been learned about the structure and regulation of the phosphoinositide specific kinases and phosphodiesterase enzymes this is a relatively new field in which we can expect many advances during the next few years.

• Chien MM, Cambier JC
• Divalent cation regulation of phosphoinositide metabolism. Naturally occurring B lymphoblasts contain a Mg2(+)-regulated phosphatidylinositol-specific phospholipase C.
• J Biol Chem. 1990; 265: 9201-7
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• Membranes isolated from normal murine B lymphocytes were found to contain a novel phosphatidylinositol (PtdIns)-specific phospholipase C (PLC) which becomes activated as the Mg2+ concentration is raised from 30 to 1000 microM. This activity, which has not been described previously in any tissue, is restricted to naturally occurring B cell blasts, i.e. it was not detected in quiescent B cells, B lymphomas, or plasmacytomas. As seen in other cell systems, B cell membranes were found to contain Mg2(+)-stimulated inositol 1,4,5-trisphosphate phosphatase activity. Although neither the inositol 1,4,5-trisphosphate phosphatase nor the PtdIns PLC activities were affected by Ca2+, B cell membranes were found to contain a Ca2(+)-stimulated phosphatidylinositol 4,5-bisphosphate (PtdInsP2) PLC activity which is activated by [Ca2+] greater than 100 nM. Based on several characteristics, it appears that the Mg2(+)- and Ca2(+)-regulated PLCs are distinct species. First, they have distinct specificity for PtdIns and PtdInsP2, respectively. Second, they have distinct tissue distribution while the Ca2(+)-regulated activity was detected in all B cells, the Mg2(+)-regulated activity is restricted to low density, natural B blasts. Third, the kinetics of activation of the enzymes is distinct; the Mg2(+)-regulated enzyme exhibits slower and less transient activation kinetics. Fourth, the activities exhibit absolute specificity in terms of activation by Mg2+ and Ca2+, i.e. the PtdIns PLC is activated only by Mg2+ and the PtdInsP2 PLC is activated only by Ca2+. Data are consistent with the possibility that Mg2+ mobilization which follows ligation of certain receptors, may play an important role in the regulation of levels of the second messenger diacylglycerol.

• Shashidhar MS, Keana JF, Volwerk JJ, Griffith OH
• Preparation and application of an affinity matrix for phosphatidylinositol-specific phospholipase C.
• Chem Phys Lipids. 1990; 56: 159-67
• Display abstract

• A non-hydrolyzable phosphonate analogue of phosphatidyl inositol, racemic myo-inosityl-(1)-5-oxa-16-trifluoroacetamidohexadecyl phosphonate, was synthesized. This phosphonate inhibited the activity of phosphatidyl inositol-specific phospholipase C (PI-PLC) from Bacillus cereus with an IC50 of approximately 10 mM. Removal of the trifluoroacetyl blocking group followed by covalent binding of the phosphonate to cyanogen bromide activated Sepharose 4B via the amino group produced an affinity matrix specific for the PI-PLC from B. cereus. This affinity matrix was used to purify the phospholipase C from a complex mixture of proteins in a single step. Competition experiments with myo-inositol in the elution medium indicated that specific binding of the enzyme to the matrix most likely involves the enzyme active site. The inositol phosphonate derivatized matrix was stable over several months in neutral and alkaline media and was used repeatedly without loss of binding capacity. These results show that affinity matrices employing myo-inositol phosphonate ligands are useful for isolation and binding studies of PI-PLC and possibly of other enzymes interacting with phosphoinositides or myo-inositol phosphate derivatives.

• Klemens MR, Sherman WR, Holmberg NJ, Ruedi JM, Low MG, Thompson LF
• Characterization of soluble vs membrane-bound human placental 5'-nucleotidase.
• Biochem Biophys Res Commun. 1990; 172: 1371-7
• Display abstract

• Three forms of 5'-nucleotidase purified from human placenta (two membrane-bound forms, one sensitive and one resistant to cleavage by phosphatidylinositol-specific phospholipase C, as well as a soluble form) had the same molecular weight before (73,000 Da) and after (56,000 Da) digestion with N-glycosidase F and showed similar amino acid compositions, N-terminal amino acid sequences, and KMs for IMP (9.6 to 11.9 microM). Thus, these three forms of 5'-nucleotidase appear to have very similar structures. The form sensitive to phosphatidylinositol-specific phospholipase C contained nearly 1 mol myo-inositol/mol of protein as determined by mass spectrometry, indicating a glycosyl phosphatidylinositol membrane anchor. Soluble 5'-nucleotidase contained a similar quantity of myo-inositol, suggesting that it was previously membrane-anchored via glycosyl phosphatidylinositol. The form resistant to phosphatidylinositol-specific phospholipase C contained less myo-inositol, leaving open the possibility of a third form of 5'-nucleotidase with a conventional transmembrane anchor.

• Bairoch A, Cox JA
• EF-hand motifs in inositol phospholipid-specific phospholipase C.
• FEBS Lett. 1990; 269: 454-6

• Volwerk JJ, Shashidhar MS, Kuppe A, Griffith OH
• Phosphatidylinositol-specific phospholipase C from Bacillus cereus combines intrinsic phosphotransferase and cyclic phosphodiesterase activities: a 31P NMR study.
• Biochemistry. 1990; 29: 8056-62
• Display abstract

• The inositol phosphate products formed during the cleavage of phosphatidylinositol by phosphatidylinositol-specific phospholipase C from Bacillus cereus were analyzed by 31P NMR. 31P NMR spectroscopy can distinguish between the inositol phosphate species and phosphatidylinositol. Chemical shift values (with reference to phosphoric acid) observed are 0.41, 3.62, 4.45, and 16.30 ppm for phosphatidylinositol, myo-inositol 1-monophosphate, myo-inositol 2-monophosphate, and myo-inositol 1,2-cyclic monophosphate, respectively. It is shown that under a variety of experimental conditions this phospholipase C cleaves phosphatidylinositol via an intramolecular phosphotransfer reaction producing diacylglycerol and D-myo-inositol 1,2-cyclic monophosphate. We also report the new and unexpected observation that the phosphatidylinositol-specific phospholipase C from B. cereus is able to hydrolyze the inositol cyclic phosphate to form D-myo-inositol 1-monophosphate. The enzyme, therefore, possesses phosphotransferase and cyclic phosphodiesterase activities. The second reaction requires thousandfold higher enzyme concentrations to be observed by 31P NMR. This reaction was shown to be regiospecific in that only the 1-phosphate was produced and stereospecific in that only D-myo-inositol 1,2-cyclic monophosphate was hydrolyzed. Inhibition with a monoclonal antibody specific for the B. cereus phospholipase C showed that the cyclic phosphodiesterase activity is intrinsic to the bacterial enzyme. We propose a two-step mechanism for the phosphatidyl-inositol-specific phospholipase C from B. cereus involving sequential phosphotransferase and cyclic phosphodiesterase activities. This mechanism bears a resemblance to the well-known two-step mechanism of pancreatic ribonuclease, RNase A.

• Bala GA, Thakur NR, Bleasdale JE
• Characterization of the major phosphoinositide-specific phospholipase C of human amnion.
• Biol Reprod. 1990; 43: 704-11
• Display abstract

• Most of the phosphoinositide-specific phospholipase C activity in human amnion at term was found to be attributable to a single isoform (Mr 85,000). Phospholipase C purified from amnion catalyzed the calcium-dependent hydrolysis of both phosphatidylinositol and phosphatidylinositol 4,5-bisphosphate. The high phospholipase C activity of amnion cells isolated at 38-41 weeks of gestation declined greater than 80% during the initial 2-5 days of culture to values characteristic of amnion tissue in early gestation. Activities of phospholipase A2 and phosphatidylinositol synthase remained essentially unaltered during this period of culture. Loss of phospholipase C activity was apparently due neither to the appearance of an inhibitor nor to the loss of an activator and most likely reflected a decrease in the amount of enzyme in amnion cells. Basal production of prostaglandin E2 (PGE2) by amnion cells also declined greatly during the period of loss of phospholipase C activity. Involvement of phospholipase C in the regulation of amnion prostaglandin production was also supported by the finding that the phospholipase C inhibitor, U-73122, potently inhibited amnion cell PGE2 production. In contrast, vasopressin, which appears to stimulate prostaglandin production in amnion cells by a phospholipase C-dependent mechanism, was equipotent in stimulating PGE2 production by amnion cells on Day 2 and Day 5 of culture, even though phospholipase C activity had declined by more than 75%. Furthermore, epidermal growth factor stimulation of PGE2 production by amnion cells appeared to be largely attributable to an increase in prostaglandin H synthase activity and did not involve an increase in phospholipase C activity.(ABSTRACT TRUNCATED AT 250 WORDS)

• Nishibe S, Carpenter G
• Tyrosine phosphorylation and the regulation of cell growth: growth factor-stimulated tyrosine phosphorylation of phospholipase C.
• Semin Cancer Biol. 1990; 1: 285-92
• Display abstract

• The significance of tyrosine phosphorylation in cell growth and transformation has been addressed. While the details of how tyrosine kinases actually function in mitogenic signal transduction is not fully understood, recent evidence suggests that phosphoinositide metabolism may have a significant role in growth factor signal transduction. Interestingly, one of phosphatidylinositol-specific phospholipase C isozymes is an excellent substrate for growth factor receptor tyrosine kinases. In this review, the current understanding of growth factor-mediated phosphoinositide metabolism and tyrosine phosphorylation of phospholipase C, is discussed.

• Rillema JA
• Possible role of phospholipase C in the regulation of cell division in normal and neoplastic cells.
• Med Hypotheses. 1989; 29: 1-4
• Display abstract

• It is proposed that a phosphatidylinositol-specific phospholipase C (PLC) enzyme may be present in abnormally high concentrations in certain cancer cells, and that the elevated activity may explain many, if not all, of the neoplastic characteristics of the cancer cells. There have thus far, been two reports in which PLC activity has been found to be elevated several fold in neoplastic cells. The products of the action of PLC on the phosphoinositides, including diglycerides and inositol phosphates, have been shown to activate the process of cell division by elevating the intracellular concentration of calcium ions and by stimulating the activity of protein kinase C. An elevated content of PLC in at least certain neoplastic cells could thus explain uncontrolled proliferative processes in those cells.

• Etindi R, Fain JN
• Insulin does not activate a phosphoinositide-specific phospholipase C in adipocytes.
• Mol Cell Endocrinol. 1989; 67: 149-53
• Display abstract

• Phosphoinositide-specific phospholipase C (PI-PLC) activity was determined in homogenates of adipocytes treated with maximal concentrations of insulin. PI-PLC activity measured using exogenous [3H]phosphatidylinositol [( 3H]PI) and exogenous [3H]phosphatidylinositol 4,5-bisphosphate [( 3H]PIP2) was not altered by prior exposure of adipocytes to insulin. It was possible to see oxytocin-induced breakdown of phosphoinositides but no effect of insulin was seen in intact adipocytes.

• Emori Y et al.
• A second type of rat phosphoinositide-specific phospholipase C containing a src-related sequence not essential for phosphoinositide-hydrolyzing activity.
• J Biol Chem. 1989; 264: 21885-90
• Display abstract

• A fourth type of rat phosphoinositide-specific phospholipase C (PLC IV) has been cloned for cDNA and sequenced. PLC IV is distinct from the other three types of rat PLC (PLC I, II, and III) with respect to primary structure and tissue distribution of its mRNAs. PLC IV contains two homologous regions included commonly in PLC I, II, and III and is most similar to PLC II (identity: 50.2%). PLC IV, in common with PLC II, has a sequence homologous to the N-terminal regulatory domains of nonreceptor tyrosine kinases of the src-family of oncogenes. Using an Escherichia coli expression system, we succeeded in producing active PLC IV in E. coli crude extracts. Various truncation experiments of the PLC IV cDNA revealed that the src-related domain is not necessary for catalytic activity while both domains homologous among PLC I-IV are essential. PLC IV is expressed in various rat tissues and abundant in spleen, suggesting that PLC IV plays a fundamental role in cellular functions such as growth and secretion.

• Wolf RA
• Cardiolipin-sensitive phospholipase C in subcellular fractions of rabbit myocardium.
• Am J Physiol. 1989; 257: 92635-92635
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• Phosphatidylinositol-specific phospholipase C was characterized in the soluble phase and in membrane fractions prepared from rabbit myocardium. Four subforms of soluble phospholipase C were identified and characterized. Activity of one subform was inhibited 80% when cardiolipin was present in substrate vesicles, whereas three subforms were stimulated 2- to 10-fold by cardiolipin. A cationic subform, molecular mass 67 kDa, was stimulated threefold when cardiolipin comprised 2% of the total phospholipid and fivefold when it comprised 12%. The major mechanism for the cardiolipin effect was a decrease in the apparent Michaelis constant (Km) of this subform for substrate. Competition experiments were consistent with binding of this subform to cardiolipin. Phospholipase C activity was present in mitochondrial, microsomal, and sarcolemmal membrane fractions that were essentially free of contamination by cytosol. Detection of membrane-associated phospholipase C was facilitated by cardiolipin. Thus rabbit myocardium contains multiple subforms of soluble phospholipase C that differ substantially in surface charge, molecular mass, and sensitivity to cardiolipin. Anionic phospholipids may be important determinants of intracellular distribution of phospholipase C in myocardial tissue.

• Toutant JP, Roberts WL, Murray NR, Rosenberry TL
• Conversion of human erythrocyte acetylcholinesterase from an amphiphilic to a hydrophilic form by phosphatidylinositol-specific phospholipase C and serum phospholipase D.
• Eur J Biochem. 1989; 180: 503-8
• Display abstract

• Each catalytic subunit in the amphiphilic dimer of human erythrocyte acetylcholinesterase (AChE) is anchored in the plasma membrane exclusively by a glycoinositol phospholipid. In contrast to erythrocyte AChEs in other mammalian species, the human enzyme is resistant to direct cleavage by phosphatidylinositol-specific phospholipase C (PtdIns-specific PLC). The resistance is due to the existence of an additional fatty acyl chain on the inositol ring which blocks the action of PtdIns-specific PLC [Roberts et al. (1988) J. Biol. Chem. 263, 18766-18775]. In this report, nondenaturing polyacrylamide gel electrophoresis was applied to permit rapid and unambiguous distinction between amphiphilic AChE, in which each catalytic subunit binds one nonionic detergent micelle, and hydrophilic AChE, which does not interact with detergent. Deacylation of human erythrocyte AChE by an alkaline treatment with hydroxylamine rendered the amphiphilic AChE susceptible to PtdIns-specific PLC with the consequent release of hydrophilic AChE. Although serum anchor-specific phospholipase D (PLD) cleaves the intact human erythrocyte AChE anchor, this treatment, as judged by nondenaturing electrophoresis, did not release hydrophilic AChE. Hydroxylamine treatment before or after PLD digestion was necessary to achieve the conversion. These observations indicate that binding of a single detergent micelle was maintained when any of the three fatty acyl or alkyl groups in the human erythrocyte AChE anchor phospholipid were retained. For proteins that can be identified following nondenaturing gel electrophoresis, these procedures provide methods both for detecting glycoinositol phospholipid anchors resistant to PtdIns-specific PLC and for indicating fatty acyl and/or alkyl chains in these anchors.

• Banno Y, Kanoh H, Nagata K, Yada Y, Nozawa Y
• Phosphoinositide-specific phospholipase C of a cloned mast cell line, mastocytoma P-815: purification and some characterization.
• Comp Biochem Physiol B. 1989; 94: 561-8
• Display abstract

• 1. Multiple forms of phosphoinositide-phospholipase C (PLC) were isolated from mastocytoma; two cytosolic forms (cPLC-I, Mr 150,000; cPLC-II, Mr 110,000) and two membrane-associated forms (mPLC-I, Mr 85,000; mPLC-II, Mr 85,000). 2. Four PLC forms differently behaved in substrate specificity and effect of GTP-binding proteins.

• Linden J, Delahunty TM
• Receptors that inhibit phosphoinositide breakdown.
• Trends Pharmacol Sci. 1989; 10: 114-20
• Display abstract

• Calcium-mobilizing receptors are believed to activate phospholipase C. Joel Linden and Therese Mary Delahunty summarize recent reports which indicate that activation of some receptors that inhibit the accumulation of Ca2+ within cells - notably receptors for adenosine, dopamine and several other neurotransmitters - can inhibit phosphoinositide metabolism. Two types of mechanism may be involved in these responses. Many instances of receptor-mediated inhibition of phosphoinositide breakdown can be detected only after a period of several minutes and may be secondary to receptor-mediated events that lower [Ca2+]i or activate certain protein kinases. In other instances the activation of receptors rapidly (within seconds) inhibits phosphoinositide breakdown, possibly via the activation of guanine nucleotide binding proteins that either directly, or by a rapid indirect action, inhibit phospholipase C. Putative mechanisms for direct and indirect regulation of phospholipase C are discussed.

• Itani Y, Oku M, Moriyama I, Ichijo M
• [Steroid block of uterine smooth muscle and regulation of phosphoinoside-specific phospholipase C]
• Nippon Heikatsukin Gakkai Zasshi. 1989; 25: 257-9

• McDonald LJ, Mamrack MD
• Purification and characterization of bovine heart phosphoinositide-specific phospholipase C: kinetic analysis of the Ca2+ requirement and La3+ inhibition.
• Biochemistry. 1989; 28: 9926-32
• Display abstract

• Bovine heart contains multiple phosphoinositide-specific phospholipase C (PIC) activities separable by ion-exchange chromatography. One PIC activity was purified to apparent homogeneity and migrated as a single band of Mr 85,000 on SDS-PAGE. The purified PIC was characterized with sonicated suspensions of either pure phosphatidylinositol 4,5-bisphosphate (PIP2) or phosphatidylinositol (PI) as substrates. At pH 7, apparent Vmax and Km values were higher for PIP2 than for PI, but the value of Vmax/Km was similar for the two substrates. PIC required Ca2+ for the hydrolysis of either PI or PIP2, and increasing free Ca2+ concentrations from 20 to 300 nM saturated PIC activity. The requirement of Ca2+ for PIC activity and the sensitivity of PIC to Ca2+ concentrations in the physiological range suggested the ion may be a cofactor. The PIC reaction mechanism was determined by two-substrate kinetic analysis; the data fit a model in which PIC contained single sites for Ca2+ and phosphoinositide, and utilized a rapid-equilibrium, random-order ternary mechanism for phosphoinositide hydrolysis. The KCa value for either PI or PIP2 hydrolysis was approximately 30 nM, suggesting resting intracellular free Ca2+ concentrations are sufficient to saturate the Ca2+ site of PIC. La3+ was used as a calcium analogue to modulate PIC activity. Low concentrations of LaCl3 (0.01-0.3 microM) inhibited PIC activity competitively with respect to calcium, consistent with a Ca2+ binding site on the enzyme.

• Homma Y, Takenawa T, Emori Y, Sorimachi H, Suzuki K
• Tissue- and cell type-specific expression of mRNAs for four types of inositol phospholipid-specific phospholipase C.
• Biochem Biophys Res Commun. 1989; 164: 406-12
• Display abstract

• The mRNA levels for four types of inositol phospholipid-specific phospholipase C (PLC) in various tissues and cell cultures have been studied by Northern analysis using cDNA probes for PLC isozyme I, II, and III [Sue, P.-G., Ryu, S.H., Moon, K.H., Sue, H.W., and Rhee, S.G. (1988) Proc. Natl. Acad. Sci. USA 85, 5419-5423 and Cell 54, 161-169], and the recently identified isozyme IV. All four types are ubiquitously expressed in rat tissues, but the levels of the mRNAs vary among tissues and cell lines. PLC-I mRNA levels are extremely high in brain and rat C6 glioma cells with lower levels in other tissues tested. PLC-II and -III have a more widespread distribution, with relatively high levels in brain, lung, spleen, thymus, and testis in the case of PLC-II, and in skeletal muscle, spleen, and testis for PLC-III. PLC-II and -III mRNAs were also detected in all cell lines examined except human promyelocytic HL60 cells. PLC-IV mRNA levels are extraordinarily high in spleen and HL60 cells. These results indicate that rat C6 glioma cells, together with most rat tissues, contain all four PLC isozymes. Other cultured cell types examined also contain two or three PLC isozymes except for HL60 cells, which contain only PLC-IV. The concomitant expression of PLC isozymes in cultured cells suggests a diverse function for PLC isozymes in single cells.

• Baldassare JJ, Henderson PA, Fisher GJ
• Isolation and characterization of one soluble and two membrane-associated forms of phosphoinositide-specific phospholipase C from human platelets.
• Biochemistry. 1989; 28: 6010-6
• Display abstract

• Two forms (mPLC-I, mPLC-II) of phosphoinositide-specific phospholipase C have been purified, 1494- and 1635-fold, respectively, from plasma membranes of human platelets. Purified mPLC-I and mPLC-II had estimated molecular weights by gel filtration and sodium dodecyl sulfate-polyacrylamide gels of 69,000 and 63,000, respectively. Two cytosolic forms (PLC-I and PLC-II) of phosphoinositide-specific phospholipase C were also resolved on a phenyl-Sepharose column. The major cytosolic form present in outdated platelets, PLC-II, was purified to homogeneity by chromatography on Fast Q-Sepharose, cellulose phosphate, heparin-agarose, phenyl-Sepharose, Superose 12, DEAE-5PW, and hydroxylapatite. Purified PLC-II had a molecular weight of 57,000 on sodium dodecyl sulfate-polyacrylamide gels. mPLC-I, mPLC-II, and PLC-II hydrolyzed both PI and PIP2. The Vmax for PIP2 hydrolysis was similar for all three forms of PLC and was approximately 5-fold greater than for PI hydrolysis. The Km for PIP2 hydrolysis was also similar for the three enzymes. In contrast, the Km for PI hydrolysis by PLC-II was 10-fold lower than by mPLC-I and mPLC-II. In addition, antibody prepared against PLC-II did not cross-react with either mPLC-I or mPLC-II. These data indicate that platelets contain membrane-associated phosphoinositide-specific phospholipases C that are distinct from at least one cytosolic form (PLC-II) of the enzyme.

• Crooke ST, Bennett CF
• Mammalian phosphoinositide-specific phospholipase C isoenzymes.
• Cell Calcium. 1989; 10: 309-23
• Display abstract

• Procaryotic and eucaryotic cells have evolved multiple pathways for communication with their external environment. The inositol 1,4,5-trisphosphate/diacylglycerol second messenger system is an example of such a signal transduction pathway which is present in multicellular eucaryotic organisms. Binding of an agonist to a specific cell surface receptor promotes rapid hydrolysis of phosphatidylinositol 4,5-bisphosphate. The pivotal enzyme for this second messenger system is phosphoinositide-specific phospholipase C which hydrolyzes phosphatidylinositol 4,5-bisphosphate to generate the two second messengers, inositol 1,4,5-trisphosphate and diacylglycerol. Recently, much progress has been made in the purification, characterization and cDNA cloning of multiple PI-PLC isoenzymes. The results of the recent studies on phosphoinositide-specific phospholipase C are reviewed.

• Choi WC, Gerfen CR, Suh PG, Rhee SG
• Immunohistochemical localization of a brain isozyme of phospholipase C (PLC III) in astroglia in rat brain.
• Brain Res. 1989; 499: 193-7
• Display abstract

• Three distinct brain isozymes of phosphoinositide-specific phospholipase C (PLC) have been previously characterized: PLC I (150 kDa), II (145 kDa) and III (85 kDa). The present immunohistochemical study of rat brain localizes PLC III to atroglia, whereas previous studies have demonstrated the neuronal localization of PLC I and II.

• Bennett CF, Balcarek JM, Varrichio A, Crooke ST
• Molecular cloning and complete amino-acid sequence of form-I phosphoinositide-specific phospholipase C.
• Nature. 1988; 334: 268-70
• Display abstract

• We report the molecular cloning and sequence of a phosphoinositide-specific phospholipase C (PI-PLC), an enzyme that is of particular interest because of its central role in cell signal transduction. The signals in question are those delivered by hormones to their cell-surface receptors that activate PI-PLC by means of a guanine nucleotide binding protein. Activation of the enzyme leads to the hydrolysis of phosphatidylinositol 4,5-bisphosphate to two second messengers, 1,2-diacylglycerol and inositol 1,4,5-trisphosphate, the second of which ultimately mobilizes internal pools of calcium. There are at least five PI-PLC isoenzymes, whose differences in structure and function are unknown. We have focused on isoenzyme I, which we have recently purified and characterized from guinea pig uterus. We have now determined the sequence of a full length complementary DNA of this isoenzyme from the rat. Although the sequence has little similarity with the only other sequenced PI-PLC isoenzyme, it has a surprising degree of similarity to thioredoxins, protein co-factors in thiol-dependent redox reactions.

• Herrero C, Cornet ME, Lopez C, Barreno PG, Municio AM, Moscat J
• Ca2+-induced changes in the secondary structure of a 60 kDa phosphoinositide-specific phospholipase C from bovine brain cytosol.
• Biochem J. 1988; 255: 807-12
• Display abstract

• The purification to homogeneity of a 60 kDa phosphoinositide-specific phospholipase C from bovine brain cytosol is reported here. This enzyme exhibits the same properties, in terms of response to Ca2+, as does the cytosolic activity in a variety of cell types. We show here that Ca2+ does not appear to modulate the binding of the enzyme to the substrate, but induces dramatic changes in its secondary structure. Therefore we suggest that a decrease in the alpha-helix content of this enzyme correlates with its ability to be activated by Ca2+.

• Homma Y
• [Heterogeneity of phosphoinositide-specific phospholipase C]
• Seikagaku. 1988; 60: 1284-90

• Homma Y, Imaki J, Nakanishi O, Takenawa T
• Isolation and characterization of two different forms of inositol phospholipid-specific phospholipase C from rat brain.
• J Biol Chem. 1988; 263: 6592-8
• Display abstract

• Two different forms of inositol phospholipid-specific phospholipase C (PLC) have been purified 2810- and 4010-fold, respectively, from a crude extract of rat brain. The purification procedures consisted of chromatography of both enzymes on Affi-Gel blue and cellulose phosphate, followed by three sequential high performance liquid chromatography steps, which were different for the two enzymes. The resultant preparations each contained homogeneous enzyme with a Mr of 85,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. One of these enzymes (PLC-II) was found to hydrolyze phosphatidyl-inositol 4,5-bisphosphate (PIP2) at a rate of 15.3 mumol/min/mg of protein and also phosphatidylinositol 4-monophosphate and phosphatidylinositol (PI) at slower rates. For hydrolysis of PI, this enzyme was activated by an acidic pH and a high concentration of Ca2+ and showed a Vmax value of 19.2 mumol/min/mg of protein. The other enzyme (PLC-III) catalyzed hydrolysis of PIP2 preferentially at a Vmax rate of 12.9 mumol/min/mg of protein and catalyzed that of phosphatidylinositol 4-monophosphate slightly. The rate of PIP2 hydrolysis by this enzyme exceeded that of PI under all conditions tested. Neither of these enzymes had any activity on phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, or phosphatidic acid. These two enzymes showed not only biochemical but also structural differences. Western blotting showed that antibodies directed against PLC-II did not react with PLC-III. Furthermore, the two enzymes gave different peptide maps after digestion with alpha-chymotrypsin or Staphylococcus aureus V8 protease. These results suggest that these two forms of PLC belong to different families of PLC.

• Katan M et al.
• Determination of the primary structure of PLC-154 demonstrates diversity of phosphoinositide-specific phospholipase C activities.
• Cell. 1988; 54: 171-7
• Display abstract

• Protein sequence analysis of a bovine brain phosphoinositide-specific phospholipase C (PI-PLC; PLC-154) has permitted the isolation of a cDNA that appears to code for this protein. Transient expression of this cDNA in COS-1 cells demonstrates that the cDNA encodes a functional phospholipase C that migrates at approximately 150,000 daltons. A transcript of approximately 7 kb is observed in RNA derived from bovine brain and a related transcript of the same size is present in certain human cell lines. Southern blot analysis indicates that one or possibly two genes hybridize with a PLC-154 probe. Regions of homology between PLC-154 and the previously described PLC-148 allow the assignment of a putative catalytic domain to the central region of PLC-154.

• Sussman JL et al.
• Purification and crystallization of a dimeric form of acetylcholinesterase from Torpedo californica subsequent to solubilization with phosphatidylinositol-specific phospholipase C.
• J Mol Biol. 1988; 203: 821-3
• Display abstract

• A dimeric form of acetylcholinesterase from Torpedo californica was purified to homogeneity by affinity chromatography subsequent to solubilization with a phosphatidylinositol-specific phospholipase C of bacterial origin. Bipyramidal crystals of the enzyme were obtained from solutions in polyethylene glycol 200. The crystals diffract to 2.0 A (1 A = 0.1 nm) resolution. They were found to be orthorhombic, space group P2221, with a = 163.4(+/- 0.2) A, b = 112.1(+/- 0.2) A, c = 81.3(+/- 0.1) A.

• Bon S, Toutant JP, Meflah K, Massoulie J
• Amphiphilic and nonamphiphilic forms of Torpedo cholinesterases: II. Electrophoretic variants and phosphatidylinositol phospholipase C-sensitive and -insensitive forms.
• J Neurochem. 1988; 51: 786-94
• Display abstract

• We report an electrophoretic analysis of the hydrophobic properties of the globular forms of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) from various Torpedo tissues. In charge-shift electrophoresis, the rate of electrophoretic migration of globular amphiphilic forms (Ga) is increased at least twofold when the anionic detergent deoxycholate is added to Triton X-100, whereas that of globular nonamphiphilic forms (Gna) is not modified. The G2a forms of the first class, as defined by their aggregation properties, are converted to nonamphiphilic derivatives by phosphatidylinositol phospholipase C (PI-PLC) and human serum phospholipase D (PLD). AChE G2a forms from electric organs, nerves, skeletal muscle, and erythrocyte membranes correspond to this type, which also exists in very small quantities in detergent-solubilized extracts of electric lobes and spinal cord. They present different electrophoretic mobilities, so that each of these tissues contains a distinct "electromorph," or two in the case of electric organs. The G2a forms of the second class (AChE in plasma, BuChE in heart), as well as G4a forms of AChE and BuChE, are insensitive to PI-PLC and PLD but may be converted to nonamphiphilic derivatives by Pronase.

• De Silva NS, Quinn PA
• Rapid screening assay for phospholipase C activity in mycoplasmas.
• J Clin Microbiol. 1987; 25: 729-31
• Display abstract

• A screening assay for phospholipase C using a chromogenic substrate incorporated into agar medium is described. The assay directly visualizes phospholipase C activity of mycoplasma lysates and membranes on agar plates, or the activity may be measured by spectrophotometry. The results from the assay confirm the presence in Ureaplasma urealyticum of phospholipase C, which is predominantly localized in the membrane fraction. The procedure has the potential to screen phospholipase C activity in other mycoplasmas and microorganisms in general.

• Manne V, Kung HF
• Characterization of phosphoinositide-specific phospholipase C from human platelets.
• Biochem J. 1987; 243: 763-71
• Display abstract

• Phosphoinositide-specific phospholipase C (PI-PLC) from human platelet cytosol was purified 190-fold to a specific activity of 0.68 mumol of phosphatidylinositol (PI) cleaved/min per mg of protein. It hydrolyses PI and phosphatidylinositol 4,5-bisphosphate (PIP2), but not phosphatidylcholine, phosphatidylserine or phosphatidylethanolamine. The enzyme exhibits an acid pH optimum of 5.5 and has a molecular mass of 98 kDa as determined by Sephacryl S-200 gel filtration. It required millimolar concentrations of Ca2+ for PI hydrolysis, whereas micromolar concentrations are optimal for PIP2 hydrolysis. Mg2+ could substitute for Ca2+ when PIP2, but not PI, was used as the substrate. EDTA was more effective than EGTA in inhibiting the basal PI-PLC activity towards PIP2. Sodium deoxycholate strongly inhibits the purified PI-PLC activity with either PI or PIP2 as substrate. Ras proteins, either alone or in the form of liposomes, have no effect on PI-PLC activity.

• Ribbes H, Plantavid M, Bennet PJ, Chap H, Douste-Blazy L
• Phospholipase C from human sperm specific for phosphoinositides.
• Biochim Biophys Acta. 1987; 919: 245-54
• Display abstract

• Human sperm lysates were incubated in the presence of 1-[14C]stearoyl-2-acyl-sn-glycero-3-phosphocholine, 1-[14C]stearoyl-2-acyl-sn-glycero-3-phosphoethanolamine or 1-[14C]stearoyl-2-acyl-sn-glycero-3-phosphoinositol. Only the latter substrate was hydrolyzed to a significant extent, with a concomitant formation of 1-[14C]stearoyl-2-acyl-sn-glycerol. Furthermore, incubation of phosphatidyl[3H]inositol under the same conditions was accompanied by the formation, in roughly equal amounts, of [3H]inositol 1-phosphate and [3H]inositol 1:2-cyclic monophosphate. Finally [32P]phosphatidylinositol 4-phosphate and [32P]phosphatidylinositol 4,5-bisphosphate were degraded into [32P]inositol 1,4-bisphosphate and [32P]inositol 1,4,5-trisphosphate, respectively. The phosphoinositide-specific phospholipase C was activated by calcium (optimal concentration 5-10 mM) and inhibited by EGTA, although endogenous calcium supported a half-maximal activity. The enzyme displayed an optimal pH of 6.0 and an apparent Km of 0.08 mM. Its specific activity was around 10 nmol/min per mg protein, which is approximately the same as that found in human blood platelets. Subcellular fractionation revealed that 55% of the enzyme was solubilized under conditions where 80% of acrosin appeared in the supernatants. The majority of the particulate phospholipase C activity (37% of total) was found in the 1000 X g pellet, which contained only 8% of total acrosin activity. Further fractionation of spermatozoa into heads and tails indicated no specific enrichment of phospholipase C activity in any of these two fractions. However, owing to a 4-fold higher protein content in the head compared to the tail fraction, it is concluded that about 80% of particulate phospholipase C activity is located in sperm head. The physiological significance of this enzyme is discussed in relation to a possible role in acrosome reaction and (or) in egg fertilization.

• Rebecchi MJ, Rosen OM
• Purification of a phosphoinositide-specific phospholipase C from bovine brain.
• J Biol Chem. 1987; 262: 12526-32
• Display abstract

• A soluble phosphoinositide-specific phospholipase C (PLC) was purified 58,000-fold from bovine brain. The enzyme, one of six distinct PLC activities detected in brain, accounted for approximately 15% of the soluble phosphatidylinositol-4,5-bisphosphate-phospholipase C (PIP2-PLC) activity in this tissue. The purification scheme included hydrophobic chromatography on phenyl-Sepharose and affinity chromatography on phosphatidylinositol-Sepharose (PI-Sepharose). The enzyme was specifically eluted from the PI-Sepharose with PI, calcium, and detergent. The purified PLC had an estimated molecular weight of 88,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and behaved as a monomeric protein during sedimentation on glycerol gradients. The enzyme required calcium for activity, exhibited a pH optimum of 6.5, and cleaved only phosphoinositides. The rates of PIP2 and phosphatidyl-4-monophosphate hydrolysis exceeded the rate of PI hydrolysis under all conditions tested. These properties are consistent with a potential role for this PLC in the early events involved in cellular calcium mobilization.

• Carter HR, Smith AD
• Resolution of the phosphoinositide-specific phospholipase C isolated from porcine lymphocytes into multiple species. Partial purification of two isoenzymes.
• Biochem J. 1987; 244: 639-45
• Display abstract

• Phospholipase C isolated from porcine mesenteric lymph node lymphocytes was distributed between the soluble and particulate fractions. Enzyme activity was found predominantly in the soluble fraction with optimal activity at pH 5.5. Gel filtration chromatography of the soluble phospholipase C revealed that it was composed of multiple species of enzyme activity. The activity associated with the particulate fraction had optimal activity at pH 7.0, as also did one of the species of soluble phospholipase C. Cellulose phosphate chromatography resolved the major soluble form into two species designated PLC-A and PLC-B. Both phenyl-Sepharose chromatography and hydroxyapatite chromatography purified these species still further. PLC-A and PLC-B demonstrated similar activities against phosphatidylinositol with a pH optimum near 5.5. The phospholipase C activities were abolished against this substrate by the addition of 1 mM-EDTA. When assayed in the presence of Ca2+-EDTA buffers providing a range of Ca2+ free concentrations, both enzymes exhibited optimal activity near 10(-3) M free Ca2+, but PLC-B was inhibited above this concentration more than PLC-A. PLC-B exhibited markedly lower activity against phosphatidylinositol 4,5-bisphosphate, suspended as liposomes of the pure phospholipid, than did PLC-A.

• Manne V
• Identification of polyphosphoinositide-specific phospholipase C and its resolution from phosphoinositide-specific phospholipase C from human platelet extract.
• Oncogene. 1987; 2: 49-54
• Display abstract

• Sonicated platelet extract, obtained after high-speed centrifugation, was fractionated by phosphocellulose chromatography, and assayed for phosphodiesteric cleavage of phosphatidylinositol (PI), phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-biphosphate (PIP2) using (i) single bilayer vesicles from total platelet membrane lipids and (ii) single bilayer vesicles from individual pure lipids. When total platelet lipids were used as the substrate, two clear-cut, but incompletely resolved, peaks of phosphoinositide-specific phospholipase C (PI-PLC) were detected; one of these activities hydrolysed PIP alone, whereas the second peak hydrolysed all three phosphoinositides. When pure phosphoinositides were used as the substrate, three distinct peaks of PI-PLC were observed; two of these peak activities correspond to the peaks when assays were conducted with total platelet membrane lipids. The third peak of activity hydrolysed PIP2 and PIP, but not PI. These results clearly show that there exist PIP/PIP2-specific PLC enzymes that do not act on PI, and escaped detection thus far because of the general practice of using PI as the substrate to monitor PI-PLC activities. I propose that these PIP/PIP2-specific PLC enzymes are the real candidates that respond to agonist occupation of receptors on the cell surface to generate the two putative second messengers inositol triphosphate and diacylglycerol.

• Schwertz DW, Halverson J, Isaacson T, Feinberg H, Palmer JW
• Alterations in phospholipid metabolism in the globally ischemic rat heart: emphasis on phosphoinositide specific phospholipase C activity.
• J Mol Cell Cardiol. 1987; 19: 685-97
• Display abstract

• The effect of global ischemia on myocardial ventricular membrane phospholipids was evaluated using a modified Langendorff preparation. Isolated rat hearts were perfused at 37 degrees C with oxygenated Krebs Ringer solution or rendered ischemic by cessation of perfusion (10 min to 3 h). Longer periods of ischemia were assessed by incubating preperfused (10 min) intact hearts in non-oxygenated Krebs (37 degrees C) for 6 to 18 h. Ischemia-induced alterations in phosphatidylinositol levels and phosphoinositide-specific phospholipase C (PI PLC) activity were assessed in detail, since inositol phospholipids and PI-PLC play putative roles in the regulation of cell function and Ca2+ homeostasis. Decreases in major membrane phospholipids (phosphatidylcholine, phosphatidylserine, cardiolipin and sphingomyelin) were demonstrated after long ischemic periods (6 to 18 h). While periods of ischemia (3 h or less) induced no change in structural phospholipids, an elevation in lysophosphatidylcholine and free fatty acids was found by 1 h. Notably a significant increase in phosphatidylinositol content and an accompanying decrease in cytosolic PI PLC activity was detected by 30 mins of ischemia. Reduced enzymic activity was not due to altered in vitro activation or deactivation of PI-PLC, to a change in the Ca2+ requirement of the enzyme, or to translocation of the enzyme from the cytosol to a membrane fraction. The isolated rat heart made globally ischemic for 30 mins under conditions described for this investigation shows signs of irreversible injury i.e. increased cell Ca2+ content and inability to initiate and maintain rhythmic contraction upon reperfusion. Therefore, it is possible that altered phosphoinositide metabolism may contribute to the evolution of ischemia-elicited irreversible cell injury.

• Taguchi R, Ikezawa H
• Properties of bovine erythrocyte acetylcholinesterase solubilized by phosphatidylinositol-specific phospholipase C1.
• J Biochem (Tokyo). 1987; 102: 803-11
• Display abstract

• The properties of acetylcholinesterase solubilized from bovine erythrocyte membrane by phosphatidylinositol (PI)-specific phospholipase C of Bacillus thuringiensis or with a detergent, Lubrol-PX, were studied. The activity of Lubrol-PX-solubilized acetylcholinesterase was broadly distributed in the fractions having Ve/Vo = 1.0-2.0 in gel filtration on a Sepharose 6B column. The intermediary fractions (Ve/Vo = 1.3-1.7) were collected as "the middle active Sepharose 6B eluate" and characterized on the basis of enzymology and protein chemistry. When this eluate was treated with PI-specific phospholipase C, the major activity peak was obtained in the later fractions with Ve/Vo = 1.75-2.0 on the same column chromatography. Lubrol-solubilized and phospholipase C-treated acetylcholinesterase preparations were different in the thermostability, the elution profiles of chromatography on Mono Q, butyl-Toyopearl and phenyl-Sepharose columns, and the affinity to phospholipid micelles. On treatment with PI-specific phospholipase C, Lubrol-solubilized acetylcholinesterase became more thermostable. The phospholipase C-treated enzyme was eluted at lower NaCl concentration from the Mono Q column than the Lubrol-solubilized enzyme. The most important difference was observed in the hydrophobicity of these two enzyme preparations. The Lubrol-solubilized enzyme shows high affinity to phospholipid micelles and hydrophobic adsorbents such as butyl-Toyopearl and phenyl-Sepharose. However, this hydrophobicity was lost when acetylcholinesterase was solubilized from bovine erythrocyte membrane by PI-specific phospholipase C. The presence of myo-inositol was confirmed in the purified preparation of acetylcholinesterase by gas chromatography (GC)-mass spectrometry (MS).(ABSTRACT TRUNCATED AT 250 WORDS)

• Bennett CF, Mong S, Wu HL, Clark MA, Wheeler L, Crooke ST
• Inhibition of phosphoinositide-specific phospholipase C by manoalide.
• Mol Pharmacol. 1987; 32: 587-93
• Display abstract

• Manoalide is a novel sesterterpenoid which has previously been shown to be a potent inhibitor of venom phospholipases A2. To determine whether manoalide inhibited other phospholipases, the sensitivity of phosphoinsitide-specific phospholipase C (PI-PLC) to inactivation by manoalide was examined using crude cytosolic PI-PLC and a PI-PLC purified to homogeneity from guinea pig uterus cytosol (PI-PLC I). Manoalide inhibited both cytosolic and purified PI-PLC I in a concentration-dependent fashion, exhibiting an IC50 of 3-6 microM. Inactivation of PI-PLC I was calcium- and pH-dependent, with greater inactivation occurring at alkaline pH. Manoalide inhibited hydrolysis of all three phosphoinositides by purified PI-PLC I. The substrate kinetics of PI-PLC I suggest that manoalide does not inhibit purified PI-PLC I by simple competitive or noncompetitive inhibition. Enzyme activity was not recovered after dialysis of manoalide-treated PI-PLC I, indicating that inactivation of PI-PLC I was irreversible. To determine whether manoalide inhibited PI-PLC in cells, the effects of manoalide on norepinephrine (NE)-stimulated phosphoinositide hydrolysis and calcium mobilization were investigated in a smooth muscle-like cell line, DDT1MF-2. Manoalide inhibited NE-induced inositol 1,4,5-trisphosphate and inositol 1-phosphate formation in a concentration-dependent manner. The IC50 for inhibition of inositol 1-phosphate formation was 1.5 microM. Manoalide also inhibited NE-induced calcium transients in DDT1MF-2 cells, exhibiting an IC50 of 2 microM. These data suggest that inhibition of PI-PLC may account, in part, for the anti-inflammatory actions of manoalide.

• Ikezawa H
• [Release of membrane-bound proteins by the action of phosphatidylinositol-specific phospholipase C (PI-specific PLase C): discovery of so-called "PI-anchoring protein"]
• Seikagaku. 1987; 59: 1334-9

• Fain JN
• Activation of phosphoinositide specific phospholipase C by ligands in the presence of guanine nucleotides.
• Prog Clin Biol Res. 1987; 249: 133-47

• Ryu SH, Suh PG, Cho KS, Lee KY, Rhee SG
• Bovine brain cytosol contains three immunologically distinct forms of inositolphospholipid-specific phospholipase C.
• Proc Natl Acad Sci U S A. 1987; 84: 6649-53
• Display abstract

• We previously reported that cytosolic fractions of bovine brain contain two immunologically distinct forms of phospholipase C (PLC), PLC-I and PLC-II. We now report the purification of another form of inositolphospholipid-specific phospholipase C from bovine brain cytosol, designated PLC-III, and the comparison of the catalytic properties of the three isozymes. Approximately 450 micrograms of pure PLC-III was obtained from 36 bovine brains, and it had a final specific activity of 30-40 mumol of phosphatidylinositol hydrolyzed per min per mg of enzyme in the presence of 0.1% deoxycholate. PLC-III exhibited an apparent Mr of 85,000 in NaDodSO4/PAGE, which is considerably smaller than the Mr of 150,000 for PLC-I and 145,000 for PLC-II. Neither of the two mixtures of monoclonal antibodies nor the rabbit polyclonal antibodies directed against either PLC-I or PLC-II cross-reacted with PLC-III. The catalytic properties of the three isozymes were studied by using small unilamellar vesicles prepared from either phosphatidylinositol (PtdIns) or phosphatidylinositol 4,5-bisphosphate (PtdInsP2) as substrates. Hydrolysis of both PtdIns and PtdInsP2 by the three enzymes was dependent on Ca2+. However, at low Ca2+ concentration, PtdInsP2 was the preferred substrate for all three enzymes. When PtdIns was the substrate, the three enzymes exhibited similar specific activities at their optimum pH, which was 4.8 for PLC-I, 5.0 for PLC-II, and 5.5 for PLC-III. But at neutral pH, the order of specific activity was PLC-III greater than PLC-II greater than PLC-I. In contrast, the order of specific activity was PLC-I greater than PLC-III greater than PLC-II for PtdInsP2 hydrolysis, which means that PLC-I is the most specific for PtdInsP2. The three enzymes were affected differently by bovine serum albumin: inhibition of PLC-I and activation of PLC-III were observed, whereas PLC-II was unaffected. This observation suggests that any putative protein effectors for PLC should be critically scrutinized.

• Hawrylak K, Stinson RA
• Phospholipase C and phosphatidylinositol phospholipase C.
• Clin Chem. 1987; 33: 337-337

• Ryu SH, Cho KS, Lee KY, Suh PG, Rhee SG
• Purification and characterization of two immunologically distinct phosphoinositide-specific phospholipases C from bovine brain.
• J Biol Chem. 1987; 262: 12511-8
• Display abstract

• We previously reported (Ryu, S. H., Cho, K. S., Lee, K. Y., Suh, P. G., and Rhee, S. G. (1986) Biochem. Biophys. Res. Commun. 141, 137-144) that cytosolic fractions of bovine brain contain two phosphoinositide-specific phospholipase C (PLC), PLC-I and PLC-II. In this paper purification procedures and properties of these two forms of enzyme are presented. The two enzymes exhibit similar substrate specificity. Both PLC-I and PLC-II catalyze the hydrolysis of phosphatidylinositol (PI), phosphatidylinositol-4-phosphate (PIP), and phosphatidylinositol-4,5-bisphosphate (PIP2). Yet, they respond differently to activators such as Ca2+ and nucleotides and to inhibitory divalent metal ions such as Hg2+ and Cd2+. In addition, they are immunologically distinct as evidenced by the fact that monoclonal antibodies directed against either enzyme do not cross-react with the other. Their activities are Ca2+ concentration-dependent. PIP and PIP2 are better substrates than PI for both PLC-I and PLC-II when the concentration of Ca2+ is in the micromolar range. Study of the effect of nucleotides, such as GTP, guanosine 5'-(3-O-thio)triphosphate, guanyl-5'-yl imidodiphosphate, and ATP, on the activities of both isozymes with PIP2 as substrate revealed that (i) in the absence of Ca2+, PLC-I activity is enhanced by 400% by either GTP or ATP. In the presence of Ca2+ (a condition in which PLC-I exhibits much higher activity), the activation factor by nucleotides is diminished to approximately 140%. (ii) without Ca2+, PLC-II activity is too low to measure with or without added nucleotides. The effect of nucleotides on PLC-II activity is trivial in the presence of Ca2+. In addition, studies on the effect of metal ions on PI hydrolysis showed that the activities of both PLC-I and PLC-II are not affected by 50 microM of Mg2+, Mn2+, Ca2+, or Ni2+. However, Hg2+, Zn2+, and Cu2+ inhibited both PLC-I and PLC-II, with PLC-II exhibiting much higher sensitivity to these metal ions than PLC-I. For example, the value of I0.5 for Hg2+ inhibition is 0.2 microM for PLC-II and 1 microM for PLC-I. Cd2+ selectively inhibits PLC-II with a I0.5 value of 5 microM. Most of these metal ions' inhibition can be overcome by either dithiothreitol or EDTA.

• Ryu SH, Cho KS, Lee KY, Suh PG, Rhee SG
• Two forms of phosphatidylinositol-specific phospholipase C from bovine brain.
• Biochem Biophys Res Commun. 1986; 141: 137-44
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• Two immunologically distinct forms of phosphoinositide-specific phospholipase C were purified to near homogeneity from bovine brain. Their molecular weights determined by SDS-PAGE are 150,000 (enzyme I) and 145,000 (enzyme II), respectively. Under a nondenaturing condition, purified enzyme I exists mainly in dimeric form and as tetramer to a small extent, while enzyme II is predominantly in monomer, to a small extent as dimer and to a very small extent as trimer. Multiple forms of phosphoinositide-specific phospholipase C in brain tissue described in the literature might be, therefore attributed to the oligomerization of the two independent forms.

• Carter HR, Bird IM, Smith AD
• Two species of phospholipase C isolated from lymphocytes produce specific ratios of inositol phosphate products.
• FEBS Lett. 1986; 204: 23-7
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• Chromatography of the soluble porcine lymphocyte phospholipase C on cellulose phosphate resolves the activity into two species. An HPLC method is described for separating the enzyme products, Ins 1,2 greater than P and Ins 1P. Use of these methods reveals that the two iso-enzymes liberate, with a high degree of reproducibility, characteristic ratios of the two products. This suggests that the amount of each product produced is an inherent property of the enzyme mechanism.

• Koch F, Thiele HG, Low MG
• Release of the rat T cell alloantigen RT-6.2 from cell membranes by phosphatidylinositol-specific phospholipase C.
• J Exp Med. 1986; 164: 1338-43
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• The mechanism by which the rat T cell alloantigen, RT-6.2, is attached to the membrane was investigated. Treatment of rat lymph node and T-hybridoma cells with phosphatidylinositol-specific phospholipase C (PI-PLC) caused a substantial reduction in the amount of RT-6.2 on the cell surface. No significant release of a rat T helper marker (visualized by the mAb W3/25) was observed in response to PI-PLC treatment. This is in sharp contrast to the effects of trypsin, which removes most of the T helper marker but had little effect on RT-6.2. SDS-PAGE analysis of the RT-6.2 released by PI-PLC indicated that the Mr was not significantly changed by this treatment. Phase separation of the released RT-6.2 in Triton X-114 showed that the PI-PLC had converted it from an amphiphilic membrane form to a water-soluble form, apparently by removing its hydrophobic membrane anchoring domain. These results strongly suggest that RT-6.2, in common with Thy-1 and several other cell surface proteins, is anchored in the membrane by the 1,2-diacylglycerol moiety of a covalently attached phosphatidylinositol molecule.

• Low MG, Carroll RC, Cox AC
• Characterization of multiple forms of phosphoinositide-specific phospholipase C purified from human platelets.
• Biochem J. 1986; 237: 139-45
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• The origin and physiological significance of the multiple Mr forms of phosphoinositide-specific phospholipase C in human platelets were investigated. The higher-Mr (400,000 and 270,000) forms of the phospholipase C were converted into the 100,000-Mr form without substantial loss of activity by incubation with a Ca2+-dependent proteinase partially purified from human platelets. These three forms of the phospholipase C were purified approx. 200-500-fold from outdated human platelet supernatants. SDS/polyacrylamide-gel electrophoresis and gel-filtration analysis suggested that the higher-Mr forms of phospholipase C were complexes of 140,000-Mr subunits, whereas the lower-Mr form consisted of a single 95,000-Mr subunit. The substrate specificity of the purified phospholipase C was investigated by using 32P-labelled polyphosphoinositide substrates purified from human platelets by a new method utilizing h.p.l.c. on an amino column. Activity against all three phosphoinositides was detected at micromolar concentrations of Ca2+; this hydrolysis was markedly stimulated by phosphatidylethanolamine and inhibited by phosphatidylcholine. Comparison of the different forms of purified phospholipase C revealed no major differences in Ca2+-sensitivity or substrate specificity. Thus, although the suggestion that the high-Mr forms of human platelet phosphoinositide-specific phospholipase C were converted into a lower-Mr form by a Ca2+-dependent proteinase has been substantiated, the physiological significance of this process remains to be determined.

• Nakanishi H, Nomura H, Kikkawa U, Kishimoto A, Nishizuka Y
• Rat brain and liver soluble phospholipase C: resolution of two forms with different requirements for calcium.
• Biochem Biophys Res Commun. 1985; 132: 582-90
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• Two forms of phospholipase C, hydrolyzing specifically inositol phospholipids, are resoluted and partially purified from rat brain as well as liver cytosol by DEAE-cellulose followed by heparin-Sepharose, Sephacryl S-400, and aminohexyl-Sepharose column chromatographies. With phosphatidylinositol as substrate, at pH 7.4 one is most active at 10(-6) M Ca2+ (Type I) whereas the other requires 10(-3) M Ca2+ (Type II). At pH 5.5 both Type I and II are active at 10(-3) M Ca2+ but essentially inactive at lower concentrations of this divalent cation. Both Type I and II hydrolyze preferentially polyphosphoinositides particularly at lower concentrations of Ca2+.

• Low MG, Carroll RC, Weglicki WB
• Multiple forms of phosphoinositide-specific phospholipase C of different relative molecular masses in animal tissues. Evidence for modification of the platelet enzyme by Ca2+-dependent proteinase.
• Biochem J. 1984; 221: 813-20
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• The Mr distribution of phosphoinositide-specific phospholipase C in the supernatants isolated from a variety of animal tissues was analysed by high-performance gel-filtration chromatography. In most tissues, at least four peaks of activity were resolved. However, different tissues showed quite marked differences in the distribution of activity between these peaks. In rat heart, lung and kidney, the predominant form had Mr approx. 90000, whereas the predominant form in brain had Mr approx. 290000. In liver, the Mr-90000 form predominated, but this tissue also contained relatively large amounts of a form of Mr approx. 150000. Phospholipase C in these tissues from other animal species gave similar distributions of activity between the peaks. In supernatants prepared from platelets sonicated in the presence of leupeptin (0.5 mM) or EGTA (20 mM), the Mr-290000 form predominated. However, when leupeptin or EGTA (inhibitors of Ca2+-dependent proteinase) was omitted from the sonication buffer, the Mr-290000 form appeared to be replaced by a form of Mr 100000. Similar changes in Mr were not demonstrated with the other tissues. These results may be relevant to the intracellular regulation of phospholipase C, since Ca2+-dependent proteolysis has been reported to occur during platelet activation.

• Nakabayashi T, Ikezawa H
• Release of alkaline phosphodiesterase I from rat kidney plasma membrane produced by the phosphatidylinositol-specific phospholipase C of Bacillus thuringiensis.
• Cell Struct Funct. 1984; 9: 247-63
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• The release of plasma-membrane-bound enzymes by phosphatidylinositol-specific phospholipase C obtained from Bacillus thuringiensis was investigated. Among the ectoenzymes of plasma membrane tested, alkaline phosphodiesterase I was released markedly from rat kidney cortex slices, in addition to alkaline phosphatase and 5'-nucleotidase. Other membrane-bound enzymes; alanine aminopeptidase, leucine aminopeptidase, dipeptidyl peptidase, leucine aminopeptidase, dipeptidyl peptidase IV, esterase and gamma-glutamyl transpeptidase could not be liberated from the treated slices. Alkaline phosphodiesterase I was released linearly from rat kidney slices with the concentration of phosphatidylinositol-specific phospholipase C, but little enzyme was released from rat liver slices. Alkaline phosphodiesterase I separated from kidney tissue with n-butanol still retained phosphatidylinositol and was transformed into a lower molecular weight form by phosphatidylinositol-specific phospholipase C. This suggests an important function for phosphatidylinositol in the binding of alkaline phosphodiesterase I to the plasma membrane of rat kidney cells. The alkaline phosphodiesterase I released from rat kidney had a molecular weight of about 240,000 and an isoelectric point (pI) of 5.4. The enzyme hydrolyzed the phosphodiester linkage of p-nitrophenyl-thymidine 5'-monophosphate at pH 8.9 and had a Km value of 0.3 mM. The enzyme was activated by Mg2+ and Ca2+, but was inhibited by EDTA. Strong inhibition took place on the addition of adenosine 5'-phosphosulfate or the nucleotide pyrophosphates, i.e., UDP-galactose and alpha, beta-methylene ATP.

• Sagawa N, Bleasdale JE, Di Renzo GC
• The effects of polyamines and aminoglycosides on phosphatidylinositol-specific phospholipase C from human amnion.
• Biochim Biophys Acta. 1983; 752: 153-61
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• The effects of polyvalent cations (polyamines and aminoglycoside antibiotics) on Ca2+-dependent phosphatidylinositol-specific phospholipase C activity of human amnion tissue were examined. In the presence of 1 mM Ca2+, the hydrolysis of phosphatidylinositol (2 mM) by phospholipase C was increased greatly (240-560% of control) by spermine (0.4 mM), spermidine (1 mM), neomycin (0.1 mM), gentamicin (0.2 mM), kanamycin (0.4 mM) and streptomycin (0.8 mM). Putrescine and cadaverine (0.1-2.0 mM), however, stimulated phospholipase C activity only slightly. The effects of spermidine, spermine and gentamicin on phospholipase C activity were characterized and found to be dependent upon the concentrations of phosphatidylinositol, Ca2+ and the particular polyvalent cation. At low concentrations of phosphatidylinositol and Ca2+ the predominant effect of polyamines and aminoglycosides was to inhibit phospholipase C activity. When the concentrations of phosphatidylinositol and Ca2+ were increased, spermidine, spermine and gentamicin stimulated phospholipase C activity. In the presence of 16 mM Ca2+, however, phospholipase C activity was maximal and was unaffected by either polyamines or aminoglycosides. At all concentrations of Ca2+ examined, the maximal stimulation of phospholipase C activity by a given polyvalent cation occurred at a fixed molar ratio of the particular polyvalent cation to phosphatidylinositol. Polyamines and aminoglycosides appeared to modulate the Ca2+ requirement for phospholipase C activity, but could not substitute completely for Ca2+. The activities of phospholipase A2, diacylglycerol lipase, monoacylglycerol lipase and diacylglycerol kinase in amnion tissue were unaffected by any of the polyvalent cations examined. It is proposed that any in vivo influences (stimulatory or inhibitory) of polyamines and aminoglycosides on amnion phospholipase C activity would depend upon the effective concentrations of Ca2+ and phosphatidylinositol.

• Chau LY, Tai HH
• Resolution into two different forms and study of the properties of phosphatidylinositol-specific phospholipase C from human platelet cytosol.
• Biochim Biophys Acta. 1982; 713: 344-51
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• Two forms of phosphatidylinositol-specific phospholipase C from human platelet cytosol were resolved by DEAE-cellulose chromatography and purified further by hydrophobic chromatography. Both forms utilized phosphatidylinositol as the best substrate. However, the enzyme did not distinguish 2-arachidonylphosphatidylinositol from 2-oleoylphosphatidylinositol although the former substrate was known to be a predominant species in human platelets. Both forms exhibited pH optimum at 7.0. Both activities were inhibited completely by 1 mM EDTA and the inhibited preparations could be restored to full activity or to 60% by free Ca2+ or Co2+, respectively, at 100 microM. Higher concentrations of either ion were inhibitory. Other metal ions were ineffective. Addition of calmodulin in the presence of Ca2+ did not show any additional effect. Both forms were inhibited comparably by various phospholipids, fatty acids and detergents, suggesting that phosphatidylinositol in membranes might be a poor substrate for the enzyme. Initiation of phosphatidylinositol breakdown through the phospholipase C pathway may require additional activator(s). A variety of anti-platelet drugs, including phenylthiazines, local anesthetics and mepacrine, were found to be potent inhibitors of the enzyme, suggesting that these drugs might inhibit platelet function by inhibiting the early phase of arachidonate release.

• Hofmann SL, Majerus PW
• Identification and properties of two distinct phosphatidylinositol-specific phospholipase C enzymes from sheep seminal vesicular glands.
• J Biol Chem. 1982; 257: 6461-9

• Fujii M, Yabu Y, Fukunaga T, Koga K
• Phospholipase C activity in egg yolk sac of Japanese quail during embryonic development.
• Int J Biochem. 1982; 14: 355-7
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• 1. The amount of lipid decreased markedly at the later stage of embryonic development, coinciding with the remarkable decrease in yolk weight. 2. Phospholipase C activity in the yolk sac appeared on the 6th day of incubation and increased markedly as incubation proceeded. 3. The enzyme showed its optimum activity at pH 9.5 and at 40 degrees C. 4. The activity was enhanced 2-fold by 20 mM CaCl2 and by 40% by 4 mM deoxycholate but inhibited by 2 mM ZnCl2 and CuSO4.

• Hetland O, Warhuus K, Giercksky KE, Aaslid R, Prydz H
• Toxicity of phospholipase C in rabbits.
• Scand J Clin Lab Invest. 1982; 42: 239-44
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• Tissue thromboplastin is the most potent physiological trigger of blood coagulation and is probably involved in the pathogenesis of several forms of intravascular coagulation. Phospholipase C from Bacillus cereus is an effective inhibitor of thromboplastin. As part of an investigation into the possible use of phospholipase C as a therapeutic or prophylactic agent in thrombosis and other forms of intravascular coagulation, we have previously studied its effect in rats. We now report on the toxicity in rabbits (along with some data on cats and monkeys). Estimated LD50 for rabbits was about 0.45-0.65 mg/kg (as compared to 1.70 mg/kg for rats). No respiratory or circulatory changes were observed, but phospholipase C caused a significant increase of several plasma enzymes in rabbits.

• Edgar AD, Freysz L
• Phospholipase activities of rat brain cytosol. Occurrence of phospholipase C activity with phosphatidylcholine.
• Biochim Biophys Acta. 1982; 711: 224-8
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• 21-day-old rat brain contains a soluble phospholipase C with the ability to hydrolyse phosphatidylcholine. This enzyme has an alkaline pH optima. The results of the DEAE-cellulose fractionation, the pH profile and the Ca2+-dependency suggest that the enzyme may be the same as that responsible for phosphatidylinositol hydrolysis. The activity of the phospholipase C is associated closely with a diacylglycerol lipase. The two enzyme activities could be separated by DEAE-cellulose fractionation, resulting in greater than 100% apparent recovery of the phospholipase C acivity. This phospholipase C activity is not the result of the back reaction of choline phosphotransferase.

• Hofmann SL, Majerus PW
• Modulation of phosphatidylinositol-specific phospholipase C activity by phospholipid interactions, diglycerides, and calcium ions.
• J Biol Chem. 1982; 257: 14359-64

• Di Renzo GC, Johnston JM, Okazaki T, Okita JR, MacDonald PC, Bleasdale JE
• Phosphatidylinositol-specific phospholipase C in fetal membranes and uterine decidua.
• J Clin Invest. 1981; 67: 847-56

• Dobias G, Krompecher E
• [A simple method for the titration of phospholipase-C]
• Orv Hetil. 1981; 122: 588-90

• Little C
• Effect of some divalent metal cations on phospholipase C from Bacillus cereus.
• Acta Chem Scand B. 1981; 35: 39-44
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• Incubation of phospholipase C from Bacillus cereus with certain divalent metal cations caused enzyme inactivation with Cu(II) being particularly effective. The inactivation arose from the reversible exchange of Zn(II) in the enzyme with the metal cations. Both zinc atoms in the enzyme exchanged rapidly with Cu(II) whereas only one exchanged spontaneously with Co(II). With lecithin substrates, CoZn-phospholipase C had a specific activity of 3.6-11.3% of that of ZnZn-phospholipase C, whereas the CoCo-enzyme was less than 1% active relative to the native enzyme. The CoZn-enzyme had the same Km value for dihexanoyllecithin as had the native enzyme, but the Vm value was markedly lower. ZnZn-, CoZn- and CoCo-phospholipase C all had very low activities towards sphingomyelin micelles, although for the CoCo-enzyme, the sphingomyelinase activity was 4-7-fold greater than for the native enzyme.

• Takenawa T, Nagai Y
• Purification of phosphatidylinositol-specific phospholipase C from rat liver.
• J Biol Chem. 1981; 256: 6769-75

• Bernheimer AW, Linder R, Avigad LS
• Stepwise degradation of membrane sphingomyelin by corynebacterial phospholipases.
• Infect Immun. 1980; 29: 123-31
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• The mechanism of in vitro synergistic lysis of sheep erythrocytes by Corynebacterium ovis and Corynebacterium equi was investigated. Hemolysis required (i) the action of phospholipase D from C. ovis, (ii) the action of an extracellular protein of C. equi, and (iii) Mg2+. Maximum lysis required imposition on the system of a fourth condition (step iv), such as chilling. Steps i, ii, and iv occur sequentially and in that order. Mg2+ functions in steps i and ii. The extracellular protein C. equi was purified to homogeneity and found to be a phospholipase C capable of hydrolyzing ceramide phosphate, phosphatidic acid, and all of the isolated major phospholipids of mammalian erythrocyte membranes. The principal features of the synergistic hemolytic system could be reproduced in experiments involving liposomes containing either sphingomyelin or ceramide phosphate and trapped [14C]glucose. We inferred that sphingomyelin of sheep erythrocytes is first converted to ceramide phosphate by C. ovis phospholipase D. On the basis of results with liposomes, we propose that the ceramide phosphate is then converted to ceramide by C. equi phospholipase C. We believe that the resulting in situ ceramide then undergoes dislocation by chilling and perhaps also by virtue of an affinity between ceramide and C. equi phospholipase C. The dislocation of ceramide presumably disorganizes the lipid bilayer sufficiently to result in cell lysis.

• Matsuzawa Y, Hostetler KY
• Properties of phospholipase C isolated from rat liver lysosomes.
• J Biol Chem. 1980; 255: 646-52
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• Phospholipase C (EC 3.1.4.3) has been identified in a soluble, delipidated protein fraction isolated from rat liver lysosomes. Lysosomal phospholipase C is active against all phospholipids tested, including phosphatidylcholine, phosphatidylinositol, phosphatidylglycerol, phosphatidylethanolamine, and phosphatidylserine. It has an acid pH optimum, does not require divalent cations, and is not inhibited by EDTA. With [1-14C]dioleoylphosphatidylcholine as the substrate, 14C-labeled monoglyceride and diglyceride are the reaction products. Monoglyceride is formed rapidly from diglyceride by a lysosomal acid lipase, although some monoglyceride may be formed directly by phospholipase C hydrolysis of lysophosphatidylcholine. The other product, phosphocholine, has been identified by its behavior during Dowex 1-formate anion exchange chromatography. This appears to be the first demonstration in mammalian systems ofa phospholipase C which is active against all phosphoglycerides.

• Low MG, Zilversmit DB
• Phosphatidylinositol distribution and translocation in sonicated vesicles. A study with exchange protein and phospholipase C.
• Biochim Biophys Acta. 1980; 596: 223-34
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• The distribution of phosphatidylinositol and phosphatidylcholine in sonicated phospholipid vesicles (phosphatidylcholine : diphosphatidylglycerol : phosphatidylinositol, 90 : 5 : 5 mol%) has been determined by the use of exchange protein from beef heart and phosphatidylinositol-specific phospholipase C from Staphylococcus aureus. Approximately 70% of the phosphatidylinositol in the sonicated vesicles was accessible to the exchange protein and 70--75% was accessible to the phospholipase C. A similar proportion (65%) of the phosphatidylcholine was accessible to the exchange protein suggesting that phosphatidylinositol was not preferentially located in either surface of the phospholipid bilayer. The rate of translocation of both phospholipids was very slow but the rate for phosphatidylcholine (t 1/2 = 4--7 days) appeared to be greater than that for phosphatidylinositol (t 1/2 = 8--60 days). Production of asymmetric vesicles by removing phosphatidylinositol from the outer surface with either exchange protein or phospholipase C did not induce rapid phospholipid translocation.

• Hostetler KY, Hall LB
• Phospholipase C activity of rat tissues.
• Biochem Biophys Res Commun. 1980; 96: 388-93

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