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Background: Nonspecific phospholipase C (NPC), which belongs to a phospholipase C subtype, is a class of phospholipases that hydrolyzes the primary membrane phospholipids, such as phosphatidylcholine, to yield sn-1, 2-diacylglycerol and a phosphorylated head-group. NPC plays multiple physiological roles in lipid metabolism and signaling in plants. To fully understand the putative roles of NPC genes in upland cotton, we cloned NPC genes from Gossypium hirsutum and carried out structural, expression and evolutionary analysis., Results: Eleven NPC genes were cloned from G. hirsutum, which were found on chromosomes scaffold269.1, D03, A07, D07, A08, D11, and scaffold3511_A13. All GhNPCs had typical phosphoesterase domains and have hydrolase activity that acts on ester bonds. GhNPCs were annotated as phospholipase C, which was involved in glycerophospholipid metabolism, ether lipid metabolism, and biosynthesis of secondary metabolites. These GhNPCs showed differential expression patterns in distinct plant tissues and in response to various types of stress (low-phosphate, salt, drought, and abscisic acid). They also had different types and numbers of cis-element. GhNPCs could be classified into four subfamilies. Four pairs of GhNPCs were generated by whole-genome duplication and they underwent purifying selection., Conclusions: Our results suggested that GhNPCs are involved in regulating key abiotic stress responses and ABA signaling transduction, and they may have various functional roles for different members under complex abiotic stress conditions. Functional divergence may be the evolutionary driving force for the retention of four pairs of duplicate NPCs. Our analysis provides a solid foundation for the further functional characterization of the GhNPC gene family, and leads to potential applications in the genetic improvement of cotton cultivars.

The physiological effects of many extracellular neurotransmitters, hormones, growth factors, and other stimuli are mediated by receptor-promoted activation of phospholipase C (PLC) and consequential activation of inositol lipid signaling pathways. These signaling responses include the classically described conversion of phosphatidylinositol(4,5)P(2) to the Ca(2+)-mobilizing second messenger inositol(1,4,5)P(3) and the protein kinase C-activating second messenger diacylglycerol as well as alterations in membrane association or activity of many proteins that harbor phosphoinositide binding domains. The 13 mammalian PLCs elaborate a minimal catalytic core typified by PLC-d to confer multiple modes of regulation of lipase activity. PLC-b isozymes are activated by Gaq- and Gbg-subunits of heterotrimeric G proteins, and activation of PLC-g isozymes occurs through phosphorylation promoted by receptor and non-receptor tyrosine kinases. PLC-e and certain members of the PLC-b and PLC-g subclasses of isozymes are activated by direct binding of small G proteins of the Ras, Rho, and Rac subfamilies of GTPases. Recent high resolution three dimensional structures together with biochemical studies have illustrated that the X/Y linker region of the catalytic core mediates autoinhibition of most if not all PLC isozymes. Activation occurs as a consequence of removal of this autoinhibition.

Plant phospholipases can be grouped into four major types, phospholipase D, phospholipase C, phospholipase A1 (PLA(1)), and phospholipase A2 (PLA(2)), that hydrolyze glycerophospholipids at different ester bonds. Within each type, there are different families or subfamilies of enzymes that can differ in substrate specificity, cofactor requirement, and/or reaction conditions. These differences provide insights into determining the cellular function of specific phospholipases in plants, and they can be explored for different industrial applications.

Pseudomonas aeruginosa-derived large extracellular protease (LepA) and hemolytic phospholipase C (PlcH) are considered to play an important role in the pathogenicity of this organism. Although bacterial growth appears to be closely related to virulence, little is known about whether LepA and PlcH participate in the growth and virulence of P. aeruginosa. In this study, we investigated whether LepA and PlcH contribute to the virulence and growth of P. aeruginosa using a wild-type strain and mutants. The growth rate of the isogenic lepA single mutant was lower than that of the wild-type strain in a minimal medium containing serum albumin or hemoglobin as the sole carbon and nitrogen source. Furthermore, the growth rate of the lepA plcH double mutant decreased greatly compared with that of the wild-type strain in a minimal medium containing erythrocytes as a sole nutrient source for growth. Thus, these results indicate that cooperation between LepA and PlcH would contribute to the utilization of erythrocytes as a sole nutrient source for the growth of P. aeruginosa. In addition, mouse infection experiments demonstrated that the virulence of the lepA and plcH single mutants was attenuated, and the numbers of the mutants were lower than the numbers of the wild-type strain in peritoneal lavage fluid and whole-blood specimens. In particular, the virulence and growth rate of the lepA plcH double mutant were markedly lower than those of the wild-type strain. Collectively, these results suggest that LepA and PlcH contribute to the in vivo virulence and growth of P. aeruginosa.

Phosphatidylcholine-hydrolyzing phospholipase C (PC-PLC) catalyzes the hydrolysis of phosphatidylcholine (PC) to generate phosphocholine and diacylglycerol (DAG). PC-PLC has a long tradition in animal signal transduction to generate DAG as a second messenger besides the classical phosphatidylinositol splitting phospholipase C (PI-PLC). Based on amino acid sequence similarity to bacterial PC-PLC, six putative PC-PLC genes (NPC1 to NPC6) were identified in the Arabidopsis genome. RT-PCR analysis revealed overlapping expression pattern of NPC genes in root, stem, leaf, flower, and silique. In auxin-treated P(NPC3):GUS and P(NPC4):GUS seedlings, strong increase of GUS activity was visible in roots, leaves, and shoots and, to a weaker extent, in brassinolide-treated (BL) seedlings. P(NPC4):GUS seedlings also responded to cytokinin with increased GUS activity in young leaves. Compared to wild-type, T-DNA insertional knockouts npc3 and npc4 showed shorter primary roots and lower lateral root density at low BL concentrations but increased lateral root densities in response to exogenous 0.05-1.0 μM BL. BL-induced expression of TCH4 and LRX2, which are involved in cell expansion, was impaired but not impaired in repression of CPD, a BL biosynthesis gene, in BL-treated npc3 and npc4. These observations suggest NPC3 and NPC4 are important in BL-mediated signaling in root growth. When treated with 0.1 μM BL, DAG accumulation was observed in tobacco BY-2 cell cultures labeled with fluorescent PC as early as 15 min after application. We hypothesize that at least one PC-PLC is a plant signaling enzyme in BL signal transduction and, as shown earlier, in elicitor signal transduction.

Fungi secrete extracellular enzymes to enable them to harvest nutrients from the environment. In the case of pathogenic fungi these enzymes can also be pathogenesis factors. Here we report the identification in fungi of a complex family of extracellular phospholipase C (PLC) enzymes, homologous to the Pseudomonas aeruginosa PLCH_PSEAE. Database searches and phylogenetic analysis showed that the PLCs clustered into two groups with different evolutionary histories. One group, subdivided into PLC-A, -B, -C and -D, was found only in aspergilli and Neosartorya fischeri. Each species only ever showed three of the four PLCs except N. fischeri which had all four PLCs plus duplicate PLC-A, -B and -C genes. Modelling studies indicated that these PLCs had mechanistic similarities to phosphoesterases and aryl sulphatases, but that they probably did not differ in substrate specificity. The second group, PLC-E, was seen in a wider range of fungi including some species of aspergilli and was always found in a head-to-head arrangement with a copper oxidase, similar to the laccases. The PLC genes appear to have arisen from separate gene transfer events from bacteria or lower eukaryotes. Thus, aspergilli have acquired PLCs twice in the course of evolution.

Due to its major role in maintaining the water-retaining properties of the epidermis, ceramide is of great commercial potentials in cosmetic and pharmaceutical industries such as in hair and skin care products. Chemical synthesis of ceramide is a costly process, and developments of alternative cost-efficient production methods are of great interest. Present study was the first attempt to perform a systematic study on the production of ceramide through enzymatic hydrolysis of sphingomyelin. Sphingomyelin hydrolysis proved to be more efficient in two-phase (water:organic solvent) system than in one-phase (water-saturated organic solvent) system. Among the screened phospholipase C, the Clostridium perfringens enzyme had the highest sphingomyelin conversion rate, with very small temperature dependence. Addition of ethanol to the system markedly enhanced the rate of ceramide formation, and a mixture of ethylacetate:hexane (50:50) was the best organic solvent tested. Other factors such as (NH(4))(2)SO(4), NaCl and CaCl(2) were also tested but excluded for further consideration. On the basis of the initial experiments, the reaction system was optimized using response surface methodology including five factors (enzyme amount, water amount, ethanol amount, reaction time and the hexane ratio of organic solvent). Water content and enzyme amount was shown to have the most significant influence on the hydrolysis reaction in the fitted quadratic model. The efficiency of sphingomyelin hydrolysis was dramatically improved through system evaluation and optimization, with the optimal conditions at 75 min reaction time, 3 Uml(-1) enzyme amount, 6% water amount, 1.8% ethanol amount and 46% hexane in ethylacetate.

A study in this issue of the Biochemical Journal by Harden and colleagues, in association with one published in the Biochemical Journal very recently [Hwang, Oh, Shin, Kim, Ryu and Suh (2005) Biochem. J. 389, 181-186], have defined a new member of the superfamily of PLC (phosphoinositide-specific phospholipase C) enzymes, PLCeta. Two isoforms, PLCeta1 and PLCeta2, and their splice variants add to the molecular diversity of PLC enzymes. The studies of PLCeta regulation suggest that at least some splice variants of PLCeta2 could be regulated by the G-protein subunits Gbetagamma. As two other families, PLCbeta and PLC, are also regulated through heterotrimeric G-proteins, this finding reveals further complexity and possible interplay between different PLC families and their regulatory networks. At this point, when it is likely that the PLCeta family completes the effort of identifying new members of this related group of PLC enzymes, I also discuss some more general concepts of PLC regulation and catalysis, and challenges awaiting their further studies.

Phospholipases C are known to be important regulators of cellular processes but may also act as virulence factors of pathogenic microbes. At least three genes in the genome of the human-pathogenic fungus Candida albicans encode phospholipases with conserved phospholipase C (Plc) motifs. None of the deduced protein sequences contain N-terminal signal peptides, suggesting that these phospholipases are not secreted. In contrast to its orthologue in Sacharomyces cerevisiae, CaPLC1 seems to be an essential gene. However, a conditional mutant with reduced transcript levels of CaPLC1 had phenotypes similar to Plc1p-deficient mutants in S. cerevisiae, including reduced growth on media causing increased osmotic stress, on media with a non-glucose carbon source, or at elevated or lower temperatures, suggesting that CaPlc1p, like the Plc1p counterpart in S. cerevisiae, may be involved in multiple cellular processes. Furthermore, phenotypic screening of the heterozygous DeltaCaplc1/CaPLC1 mutant showed additional defects in hyphal formation. The loss of CaPLC1 cannot be compensated by two additional PLC genes of C. albicans (CaPLC2 and CaPLC3) encoding two almost identical phospholipases C with no counterpart in S. cerevisiae but containing structural elements found in bacterial phospholipases C. Although the promoter sequences of CaPLC2 and CaPLC3 differed dramatically, the transcriptional pattern of both genes was similar. In contrast to CaPLC1, CaPLC2 and CaPLC3 are not essential. Although Caplc2/3 mutants had reduced abilities to produce hyphae on solid media, these mutants were as virulent as the wild-type in a model of systemic infection. These data suggest that C. albicans contains two different classes of phospholipases C which are involved in cellular processes but which have no specific functions in pathogenicity.

PLC (phospholipase C) plays an important role in intracellular signal transduction by hydrolysing phosphatidylinositol 4,5-bisphosphate, a membrane phospholipid. To date, 12 members of the mammalian PLC isoforms have been identified and classified into five isotypes beta, gamma, delta, epsilon and zeta, which are regulated by distinct mechanisms. In the present study, we describe the identification of a novel PLC isoform in the brains of human and mouse, named PLC-eta, which contains the conserved pleckstrin homology domain, X and Y domains for catalytic activity and the C2 domain. The first identified gene encoded 1002 (human) or 1003 (mouse) amino acids with an estimated molecular mass of 115 kDa. The purified recombinant PLC-eta exhibited Ca2+-dependent catalytic activity on phosphatidylinositol 4,5-bisphosphate. Furthermore, molecular biological analysis revealed that the PLC-eta gene was transcribed to several splicing variants. Although some transcripts were detected in most of the tissues we examined, the transcript encoding 115 kDa was restricted to the brain and lung. In addition, the expression of the 115 kDa protein was defined in only nerve tissues such as the brain and spinal cord. In situ hybridization analysis with brain revealed that PLC-eta was abundantly expressed in various regions including cerebral cortex, hippocampus, zona incerta and cerebellar Purkinje cell layer, which are neuronal cell-enriched regions. These results suggest that PLC-eta may perform fundamental roles in the brain.

Phospholipase C (PLC)-delta1 protein appears to accumulate aberrantly in Alzheimer's disease brains and its expression is reported to be induced by overstimulation of N-methyl-D-aspartate (NMDA) receptor, but there is little knowledge on its physiological role. To clarify this, we examined the expression profile of PLC-delta1 in primary cultured rat cortical neurons treated with NMDA or peroxynitrite, in comparison with those of PLC-beta1 and -gamma1, the overexpression of both of which protects cells from oxidative stress. Overstimulation of NMDA receptor decreased and increased the expression of PLC-beta1 and -delta1, respectively, but did not affect that of PLC-gamma1, in the neurons. The viability of neurons decreased depending on the period of treatment with S-nitroso-N-acetyl D,L-penicillamine (SNAP), there being a significant decrease on 9 h treatment. On examination of the expression profiles of PLC isozymes after treatment of neurons with SNAP, PLC-beta1 was found to be increased after 1h treatment and decreased after 9 h treatment, while PLC-delta1 was significantly increased, especially after 5 h treatment. Peroxynitrite treatment caused a dose-dependent decrease in the viability of neurons, and expression of PLC-beta1 was increased by a nontoxic level of peroxynitrite and decreased by a toxic level of it, while that of PLC-delta1 was increased by a sublethal level of it. These findings suggested that induction of PLC-beta1 might protect neurons from oxidative stress, but that of PLC-delta1 might have the opposite role, although both isozymes responded to oxidative stress.

The conceptual segregation of G protein-stimulated cell signaling responses into those mediated by heterotrimeric G proteins versus those promoted by small GTPases of the Ras superfamily is no longer vogue. PLC-epsilon, an isozyme of the phospholipase C (PLC) family, has been identified recently and dramatically extends our understanding of the crosstalk that occurs between heterotrimeric and small monomeric GTPases. Like the widely studied PLC-beta isozymes, PLC-epsilon is activated by Gbetagamma released upon activation of heterotrimeric G proteins. However, PLC-epsilon markedly differs from the PLC-beta isozymes in its capacity for activation by Galpha(12/13) - but not Galpha(q) -coupled receptors. PLC-epsilon contains two Ras-associating domains located near the C terminus, and H-Ras regulates PLC-epsilon as a downstream effector. Rho also activates PLC-epsilon, but in a mechanism independent of the C-terminal Ras-associating domains. Therefore, Ca(2+) mobilization and activation of protein kinase C are signaling responses associated with activation of both H-Ras and Rho. A guanine nucleotide exchange domain conserved in the N terminus of PLC-epsilon potentially confers a capacity for activators of this isozyme to cast signals into additional signaling pathways mediated by GTPases of the Ras superfamily. Thus, PLC-epsilon is a multifunctional nexus protein that senses and mediates crosstalk between heterotrimeric and small GTPase signaling pathways.

In this report we describe the 1,500-fold purification and characterization of the haemolytic phospholipase C (PLC) of Pseudomonas aeruginosa, the paradigm member of a novel PLC/phosphatase superfamily. Members include proteins from Mycobacterium tuberculosis, Bordetella spp., Francisella tularensis and Burkholderia pseudomallei. Purification involved overexpression of the plcHR1,2 operon, ion exchange chromatography and native preparative polyacrylamide gel electrophoresis. Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry confirmed the presence of two proteins in the purified sample with sizes of 17,117.2 Da (PlcR2) and 78,417 Da (PlcH). Additionally, liquid chromatography electrospray mass spectrometry (LCMS) revealed that PlcH and PlcR2 are at a stoichiometry of 1 : 1. Western blot analysis demonstrated that the enzyme purifies as a heterodimeric complex, PlcHR2. PlcHR2 is only active on choline-containing phospholipids. It is equally active on phosphatidylcholine (PC) and sphingomyelin (SM) and is able to hydrolyse plasmenylcholine phospholipids (plasmalogens). Neither PlcHR2 nor the M. tuberculosis homologues are inhibited by D609 a widely used, competitive inhibitor of the Bacillus cereus PLC. PlcH, PlcR2, and the PlcHR2 complex bind calcium. While calcium has no detectable effect on enzymatic activity, it inhibits the haemolytic activity of PlcHR2. In addition to being required for the secretion of PlcH, the chaperone PlcR2 affects both the enzymatic and haemolytic properties of PlcH. Inclusive in these data is the conclusion that the members of this PC-PLC and phosphatase family possess a novel mechanism for the recognition and hydrolysis of their respective substrates.

Upon fertilisation by sperm, mammalian eggs are activated by a series of intracellular Ca(2+) oscillations that are essential for embryo development. The mechanism by which sperm induces this complex signalling phenomenon is unknown. One proposal is that the sperm introduces an exclusive cytosolic factor into the egg that elicits serial Ca(2+) release. The 'sperm factor' hypothesis has not been ratified because a sperm-specific protein that generates repetitive Ca(2+) transients and egg activation has not been found. We identify a novel, sperm-specific phospholipase C, PLC zeta, that triggers Ca(2+) oscillations in mouse eggs indistinguishable from those at fertilisation. PLC zeta removal from sperm extracts abolishes Ca(2+) release in eggs. Moreover, the PLC zeta content of a single sperm was sufficient to produce Ca(2+) oscillations as well as normal embryo development to blastocyst. Our results are consistent with sperm PLC zeta as the molecular trigger for development of a fertilised egg into an embryo.

The two studies presented here were done to determine the prevalence of the alpha, beta, epsilon and enterotoxin genes and the novel beta2 toxin gene of Clostridium perfringens in neonatal or pre-weaned piglets with diarrhoea or necrotic enteritis. All C. perfringens isolates were positive for the alpha and negative for the epsilon and enterotoxin gene, implying that only non-enterotoxigenic type A and C strains were detected. The most important findings were the relatively high prevalence of the beta2 toxin gene in isolates from diarrhoeic piglets in both studies, and, in one of the two studies, absence of strains with only the alpha and beta toxin gene. These data are supportive for the suggestion of a causal relationship of beta2 toxin-producing strains with digestive tract diseases in piglets.

Bacterial phospholipases C are known to act on biomembranes, since they can cleave the phosphodiester linkage between the polar head and the hydrophobic moiety of each phospholipid in these membranes. These enzymes have been classified into three groups; phosphatidylcholine (PC)-, sphingomyelin (SM)- and phosphatidylinositol (PI)-degrading phospholipases C. Enzymatic properties and toxicities of these phospholipases C are reviewed, in relation to author's research. Studies on the hemolytic phospholipases of Clostridium sp., Bacillus cereus etc., revealed that hydrolysis of choline-containing phospholipids such as PC and SM was responsible for the hemolysis of mammalian erythrocytes by these enzymes in the presence of Ca2+ and/or Mg2+. Also, the studies on a structure-activity correlation of SM-hydrolyzing phospholipase C from B. cereus disclosed the similarity of active sites between this enzyme and bovine pancreatic DNase I. By action of PI-degrading phospholipases C, several membrane proteins such as alkaline phosphatase, 5'-nucleotidase, VSG (protozoal surface glycoprotein) etc., were shown to be released from the plasma membranes of eucaryotic cells. From structural analysis, these proteins have been revealed to be glycosylphosphatidylinositol (GPI)-anchored proteins bound to the plasma membranes with carboxyl terminal-attached glycolipid. Biochemistry and molecular biology of GPI-anchored proteins, including the structures and biosynthetic routes of GPI glycolipids as well as the process of GPI attachment to proteins, requirements of C-terminal signal peptide for the protein modification by GPI, and distribution of GPI-anchored proteins in living world, are described in relation to our studies.

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.

Recent structural studies of mammalian phosphoinositide-specific phospholipase C (PI-PLC) have begun to shed light on the mechanism whereby this family of effector enzymes is able to hydrolyze phospholipid substrates to yield second messengers. PI-PLC isozymes employ a variety of modules (PH domain, EF-hand domain, SH2 domain, SH3 domain and C2 domain) that are common in proteins involved in signal transduction to reversibly interact with membranes and protein components of the signalling pathways.

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.

In mature rat brain, PLC beta 3 mRNA was detected weakly only in the pituitary gland and the cerebellar Purkinje and granule cells whereas PLC beta 4 mRNA was expressed intensely in the olfactory mitral cells, thalamic nuclei, medial habenula, pituitary gland and cerebellar Purkinje and granule cells. The beta 4 mRNA was also detected discretely in large neurons of presumably cholinergic nature, scattered rather evenly throughout the caudate putamen and diagonal band, although no significant expression was seen in the medium spiny neurons in the caudate putamen, and the hippocampal pyramidal cells and dentate granule cells. PLC gamma 2 mRNA was localized only in the Purkinje and granule cells in the vermal portions of lobules IX and X of the cerebellum, and in the adenohypophysis.

Epidermal growth factor (EGF) binding increases in late-gestational rat kidney and then falls toward basal adult levels postnatally during the 1st wk. We report that the increase in EGF binding is accompanied by an increase in EGF receptor (EGFR) protein and activation of EGFR tyrosine kinase. Multiple proteins were endogenously tyrosine phosphorylated in kidney membranes from fetal rats, and the phosphorylation pattern was similar in rats ranging from 16 to 21 days of gestation. Tyrosine phosphorylation was, however, almost undetectable in 12-wk adult rat kidneys (controls). Among the phosphoproteins in fetal kidney, a prominent 170-kDa protein was identified as EGFR. Endogenous tyrosine phosphorylation of EGFR (reflecting receptor activation) was 30-fold higher in fetal kidney membranes than in adult (3- to 7-fold higher when adjusted for differences in EGF binding or EGFR protein content). The EGFR substrate, phospholipase C-gamma 1, was tyrosine phosphorylated in fetal kidneys but not adult, and a greater proportion was membrane-associated in fetal kidneys, consistent with activation of phospholipase C-gamma 1. Thus EGFR tyrosine kinase activity is increased in late-gestational rat kidney. Induction and activation of EGFR may mediate perinatal renal cell growth and development.

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.