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Query: EC:3.1.4.3 (phospholipase C)
 18,461 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Three phospholipase C isozymes (PLC-I, II, and III) have been purified from bovine brain. Here, phospholipase C-related cDNA clones corresponding to PLC-I and PLC-III were isolated from a rat brain lambda gt11 expression cDNA library using specific monoclonal antibodies and sequenced. Each of them encodes a distinct polypeptide with a calculated molecular mass of 138,225 (PLC-I) and 85,840 (PLC-III). Comparison of these two with the sequence of another isozyme PLC-II (Mr = 148,431) that we have previously characterized revealed a low overall sequence homology. Nevertheless, a significant amino acid sequence similarity between the three enzymes was found in two regions, one of about 150 amino acids and the other of about 120 amino acids. The two conserved domains were separated by a variable region. The variable region sequence of PLC-II is relatively long and has recently been shown to contain regions homologous to the noncatalytic domain of the nonreceptor tyrosine kinases. Those of PLC-I and III were short and appeared to be unrelated to these tyrosine kinases. The physiological implications of the multiple species of PLC enzymes are discussed.
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PMID:Cloning and sequence of multiple forms of phospholipase C. 339 Aug 63

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.
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PMID:Bovine brain cytosol contains three immunologically distinct forms of inositolphospholipid-specific phospholipase C. 347 95

Sheep seminal vesicles contain two immunologically distinct phospholipase C (PLC) enzymes that can hydrolyze phosphatidylinositol (PI) (Hofmann, S.L., and Majerus, P.W. (1982) J. Biol. Chem. 257, 6461-6469). One of these enzymes (PLC-I) has been purified to homogeneity; the second (PLC-II) has been purified 2600-fold from a crude extract of seminal vesicles. In the present study we have compared the ability of these purified enzymes to hydrolyze PI, phosphatidylinositol 4-phosphate (PI-4-P), and phosphatidylinositol 4,5-diphosphate (PI-4,5-P2). Using radiolabeled substrates in small unilamellar phospholipid vesicles of defined composition, the two enzymes were found to hydrolyze all three of the phosphoinositides. Hydrolysis of all three phosphoinositides by both enzymes was stimulated by Ca2+; however, in the presence of EGTA only the polyphosphoinositides were hydrolyzed. The two enzymes displayed substrate affinities in the order PI greater than PI-4-P greater than PI-4,5-P2, and maximum hydrolysis rates in the order PI-4,5-P2 greater than PI-4-P greater than PI. When present in the same vesicles, PI and the polyphosphoinositides competed for a limiting amount of either enzyme. Inclusion of phosphatidylcholine into vesicles containing the phosphoinositides resulted in greater inhibition of PI hydrolysis than polyphosphoinositide hydrolysis. When all three phosphoinositides were present in vesicles mimicking the cytoplasmic leaflet of cell membranes, there was preferential hydrolysis of the polyphosphoinositides over PI. We conclude that a single phospholipase C can account for the hydrolysis of all three phosphoinositides seen during agonist-induced stimulation of secretory cells. The cytoplasmic Ca2+ concentration and phospholipid composition of the membrane, however, may influence the relative rate of hydrolysis of the three phosphoinositides.
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PMID:Hydrolysis of polyphosphoinositides by purified sheep seminal vesicle phospholipase C enzymes. 609 Apr 45

Two forms (I and II) of phosphoinositide-specific phospholipase C (PLC) were purified from the cytosol of bovine iris sphincter by sequential chromatography on DEAE-Sepharose, EAH-Sepharose, heparin-Sepharose, Sephacryl S-200 gel filtration and Mono Q HR columns. The final step resulted in specific activities of PLC-I and PLC-II of 4.3 and 5.9 mumol of phosphatidylinositol (PI) cleaved/min per mg of protein, which represented up to 295-fold purification compared with that of the starting supernatant. The purified enzymes were further investigated for the presence of isoenzymes and characterized for molecular mass, substrate specificity, pH, Ca2+ requirements and kinetic parameters. Using monoclonal antibodies, PLC-I was identified as PLC-delta 1. The apparent molecular mass of PLC-I as determined by SDS/PAGE and gel filtration was 85 kDa. PLC-II contained an apparently invisible protein band that reacted with the antibody against PLC-gamma 1, and a major 109 kDa protein band that was not recognized by any of the PLC monoclonal antibodies. Further purification of PLC-II by size-exclusion h.p.l.c. resulted in elution of the enzyme activity as a single peak which corresponded to 109 kDa position. Again, this PLC activity was not recognized by any of the PLC monoclonal antibodies. However, the 109 kDa protein activity was recognized by a polyclonal antibody raised against a rat PLC-gamma 1 fragment (amino acids 1272-1287), thus suggesting that this protein is a proteolytic product of PLC-gamma 1. PLC-delta 1 and PLC-gamma 1 were identified in the supernatant fraction and PLC-beta 1 in the membrane fraction of the iris sphincter. Although immunologically different, the catalytic properties of PLC-I and PLC-II were quite similar. The Vmax and Km values for phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis were three to five times greater than those for PI hydrolysis. Both forms preferred PIP and PIP2 over PI and both were inactive against phosphatidylcholine. With PIP2 as substrate, the optimal pH values for PLC-I and PLC-II were 6.5 and 7.5 respectively. Unlike PIP2, PI hydrolysis by both forms was dependent on the presence of free Ca2+. The maximal hydrolysis of PI and PIP2 by both forms occurred at 200 and 5 microM Ca2+ respectively. Incubation of the purified enzymes with the catalytic subunit of protein kinase A (PKA) and [gamma-32P]ATP resulted in increased phosphorylation of PLC-I and PLC-II, but it had no inhibitory effect on their enzyme activities.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Purification and characterization of phosphoinositide-specific phospholipase C from bovine iris sphincter smooth muscle. 838 Sep 92

Activation of phospholipase C (PLC) coupled to phosphoinositide (PtdIns) hydrolysis occurs through one of the two pathways. One of the major pathways for the neurotransmitter signaling involves phosphoinositide (PtdIns) specific and G-protein dependent PLC-beta, which stimulates the formation of inositol triphosphate (IP3) and inositol tetraphosphate (IP4). Another pathway through the stimulation of calcium influx can directly activate all of the PLC isozymes. At least three isozymes of PLC have been characterized in the brain; PLC-A (alpha), PLC-I (beta) and PLC-II (gamma), which are shown to be localized differentially in brain regions. Muscarinic-cholinergic signals are mediated in large part through the hydrolysis of PtdIns by PLC. To investigate changes in muscarinic coupling to PLC during aging, we examined carbachol stimulated and calcium stimulated PtdIns hydrolysis in cerebral cortical membranes in young, middle aged and old rats. In order to determine whether PtdIns hydrolysis changes correspond to PLC isozyme expression in these animals, we examined three subtypes of PLC mRNA expression in brain sections of young and old rats using in situ hybridization technique. Our study indicated decreased carbachol-induced PLC activity in the cerebral cortex and, in contrast, increased PLC-beta mRNA in the frontal cortex and superficial cortical layer of aged rats. PLC-alpha mRNA was decreased in hippocampal regions of older rats. These studies suggest that during aging there is an uncoupling of muscarinic stimulated PtdIns hydrolysis, which is accompanied by an increased PLC-beta mRNA and decreased PLC-alpha mRNA that may represent compensatory changes in PLC expression.
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PMID:Age-related loss of cholinergic-muscarinic coupling to PLC: comparison with changes in brain regional PLC subtypes mRNA distribution. 872 Aug 70


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