Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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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)

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

In order to determine which portion of phosphoinositide-specific phospholipase C (PLC)-beta 1 is required for activation by G alpha q, a series of specific deletions and truncations of PLC-beta 1 cDNA were prepared. After transfection of COS-7 cells with these cDNA clones, the activity and localization of the expressed proteins were determined. Specific deletions in the C-terminal end of the protein did not lead to loss of intrinsic enzymatic activity but did result in loss of the ability to be activated by G alpha q. The region required for activation was localized to the amino acid sequence corresponding to residues 903-1142 of PLC-beta 1. This region was further subdivided into two sequences; one extending from residues Thr-903 to Gln-1030 that was required for particulate fraction association as well as for activation by G alpha q and the other extending from residues Gln-1030 to Leu-1142 that was required for interaction with G alpha subunits. These results were confirmed by the observation that the C-terminal portion of PLC-beta 1, when co-expressed with the muscarinic acetylcholine receptor type 1 or the alpha 1C-adrenergic receptor in COS-7 cells, markedly inhibited ligand-induced release of inositol phosphates. In an in vitro system, two peptides derived from the G-protein interaction region at the C terminus were found to inhibit the guanosine 5'-3-O-(thio)triphosphate-dependent activation of PLC-beta 1 by G alpha q. This further localized the sites on PLC-beta 1 which are involved in interaction with G-protein alpha subunits.
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PMID:Identification of critical regions on phospholipase C-beta 1 required for activation by G-proteins. 838 37

The present study was performed to determine whether transforming growth factor beta 1 (TGF beta 1) and tissue renin-angiotensin (R-A) system are involved in hypertrophic cardiomyopathy. Cardiomyopathic Syrian hamsters (Bio 14.6) aged 4 and 20 weeks were used as a model of hypertrophic cardiomyopathy and compared with age-matched F1 beta Syrian hamsters. Total RNA was extracted from the left ventricle, and the m-RNA expressions of TGF beta 1 and angiotensinogen (ATN) were examined by Northern blotting or Ribonuclease Protection Assay (RPA). The activity of angiotensin-converting enzyme (ACE) was assayed by the modified method of Tess, using crude membrane fraction prepared from left ventricle. The effect of angiotensin II (A II) on phosphatidylinositol (PI) metabolism was evaluated by the PI -or PIP2 (phosphatidylinositol 4,5-bis phosphate)-specific phospholipase C (PLC), which releases inositol-1,4,5-triphosphate (I P3) and diacylglycerol (DAG) in cardiac myocytes. The m-RNA expressions of TGF beta 1 and ATN were detected in each group of Syrian hamsters (BIO14.6 and F1 beta). TGF beta 1 m-RNA expression was markedly increased in BIO14.6 compared with F1 beta at the age of 4 weeks, and was more intensified at the age of 20 weeks, while no significant difference was demonstrated in the ATN m-RNA expression. ACE activity in the left ventricle was enhanced in 20 week-old BIO14.6 compared with age-matched F1 beta. The activities of PI- and PIP2-specific PLC were enhanced in 20 week-old BIO14.6 in response to A II stimulation. DAG and IP3, which are second messengers and activate protein kinase C. were significantly released from the cardiac myocytes of 20 week-old BIO14.6. These results suggest that the increase in expression of TGF beta 1 gene in the left ventricle may induce cardiac hypertrophy in BIO14.6, and that the exaggerated response of phosphatidylinositol metabolism to A II and the increased activity of ACE in cardiac tissue R-A system may lead to the development of cardiac hypertrophy.
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PMID:[Tissue factors contributing to cardiac hypertrophy in cardiomyopathic hamsters (BIO14.6): involvement of transforming growth factor-beta 1 and tissue renin-angiotensin system in the progression of cardiac hypertrophy]. 838 86

The beta gamma subunits of guanine nucleotide-binding proteins (G proteins) have been shown to activate unidentified phospholipase C (PLC) isozymes (Camps, M., Hou, C., Sidiropoulos, D., Stock, J. B., Jakobs, K. H., and Gierschik, P. (1992) Eur. J. Biochem. 206, 821-831; Blank, J. L., Brattain, K. A., and Exton, J. H. (1992) J. Biol. Chem. 267, 23069-23075). To identify these target PLC isozymes, we measured the effect of bovine brain G protein beta gamma subunits on PLC-beta 1, PLC-beta 2, PLC-beta 3, PLC-gamma 1, and PLC-delta 1 activity by reconstituting purified protein components with lipid vesicles containing [3H]phosphatidylinositol 4,5-bisphosphate (PtdIns 4,5-P2). A nearly saturating concentration of beta gamma produced 2.5-, 4-, 8.5-, and 2-fold increases in PLC-beta 1, PLC-beta 2, PLC-beta 3, and PLC-delta 1 activity, respectively, and no activation of PLC-gamma 1, in the presence of 0.2 microM free Ca2+. The beta gamma-dependent activation of the PLC-beta isozymes does not appear to be the result of increased affinity of the enzymes for Ca2+. The beta gamma-dependent PLC activation could be reversed by addition of the GDP-bound form of the alpha subunit of G(o). The alpha subunits of Gq class G proteins have been shown to activate PLC-beta isozymes in the order of PLC-beta 1 > or = PLC-beta 3 >> PLC-beta 2, which differs from the order of PLC-beta 3 > PLC-beta 2 > PLC-beta 1 for beta gamma-dependent activation. Furthermore, the half-maximal concentration of beta gamma (25 nM) required to activate PLC-beta 3 is much higher than that of Gq alpha subunits (0.6 nM) required to activate PLC-beta 1. These results suggest that the extracellular signals that induce the dissociation of G(o) or Gi, the heterotrimeric G proteins abundant in brain, should enhance the hydrolysis of PtdIns 4,5-P2 in brain primarily through activation of PLC-beta 3 (PLC-beta 2 is not detectable in brain). However, signals that activate the less abundant Gq class heterotrimers should result in the activation primarily of PLC-beta 1 and PLC-beta 3 by the corresponding alpha subunits.
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PMID:Activation of phospholipase C isozymes by G protein beta gamma subunits. 838 16

We report here further characterization of the Chinese hamster lung fibroblast mutant D1-9b displaying elevated agonist-induced phosphatidylinositol (PI) turnover responses relative to CCL39, the parental cell line (Rath, H. M., Doyle, G. A. R., and Silbert, D. F. (1989) J. Biol. Chem. 264, 13387-13390). These differences in PI turnover responses are further enhanced by long term (24 h) pretreatment of cells with the phorbol ester 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA). Short term pretreatment (15 min) with PMA attenuates the agonist-induced PI turnover response of both wild type and mutant D1-9b cell lines to that of unstimulated controls, suggesting that PMA-induced desensitization responses in D1-9b are intact. Both cytosolic and membrane/particulate fractions prepared from mutant D1-9b have decreased phospholipase C (PLC) activities relative to wild type when assayed with either of four different PI/phosphatidylinositol 4,5-bisphosphate-mixed micelle/vesicle substrate combinations. Thermolability studies, Mono Q anion-exchange chromatography, and Western blot studies identified the source of cytosolic PLC deficiency in D1-9b as being due to the absence of PLC delta 1, one of at least two PLC isozymes previously shown to be in wild type CCL39 cytosol (Rath, H. M., Fee, J. A., Rhee, S. G., and Silbert, D. F. (1990) J. Biol. Chem. 265, 3080-3087). We also report here the presence of two peaks of PLC activities (i.e. peaks mA and mB) following Mono Q chromatography of wild type CCL39 membrane/particulate extracts; membrane/particulate extracts prepared from mutant D1-9b are missing the first of these two peaks of activities (peak mA). Immunoblot analysis confirms the presence of PLC beta 1, but not PLC gamma 1 or PLC delta 1, in peak mB. Comparisons are made between D1-9b and the previously characterized mutant D1-6b, which displays diminished agonist-induced PI turnover responses yet in vitro biochemical deficiencies similar to those of mutant D1-9b. Somatic cell hybridization experiments suggest that the defects found in mutant D1-9b are codominant relative to wild type phenotype and that mutant D1-9b belongs to the same genetic complementation group as mutant D1-6b. Possible explanations to explain these different agonist-induced responses in light of the two mutants similar in vitro biochemical deficiencies are addressed.
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PMID:Characterization of a second hamster lung fibroblast mutant with defects in phosphatidylinositol-specific phospholipase C. 838 94

Specific antisera were produced to peptides representing the carboxyl termini of three subtypes of phosphatidylinositol-specific phospholipase C (PIPLC) beta which have been identified by isolation of cDNAs (Kriz, R., Lin, L., Sultzman, L., Ellis, C., Heldin, C., Pawson, T., and Knopf, J. (1990) Ciba Found. Symp. 150, 112-127). Screening with the antisera indicates that PIPLC beta 3 is present in a variety of cell lines and rat tissues, whereas the distribution of PIPLC beta 1 and beta 2 is more restricted. A combination of conventional and immunoaffinity chromatographic techniques was used to purify PIPLC beta 1 and beta 3 from rat brain membranes. PIPLC beta 2 was purified from cytosol of HL60 cells. All three subtypes were activated by purified G protein alpha q/11 subunits with the following relative efficacies: PIPLC beta 3 > or = PIPLC beta 1 >> PIPLC beta 2. All three PIPLC subtypes were also activated by G protein beta gamma subunits with varying efficacies. The presence of beta gamma subunits depressed the ability of alpha q/11 to activate PIPLC beta 1 and beta 3 at low Mg2+ concentrations (1 mM). At higher concentrations of Mg2+ (2 mM or greater), activation of PIPLC beta 3, but not PIPLC beta 1, by beta gamma and alpha q/11 became additive. PIPLC beta 3 was activated by alpha q/11 even in the presence of a saturating concentration of beta gamma subunits. This indicates that there are separate sites for interaction of PIPLCs with G protein subunits and that this interaction differs depending on the enzyme subtype and the concentration of Mg2+.
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PMID:Regulation of purified subtypes of phosphatidylinositol-specific phospholipase C beta by G protein alpha and beta gamma subunits. 838 2

The phosphoinositide signal transduction pathway mediates important processes in intestinal physiology, yet the key enzyme, phosphoinositide-specific phospholipase C (PI-PLC), is not well-characterized in the colon. PI-PLC activity was examined in rat colonic membranes using exogenous [3H]phosphatidylinositol 4,5-bisphosphate (PIP2) as substrate, and beta-glycerophosphate to suppress degradation of substrate or product. The activity of membrane PI-PLC increased 6-fold with the addition of alamethicin, and a further 2-3-fold enhancement was observed with 10 microM guanosine 5'-[gamma-thio]triphosphate (GTP[S]), suggesting the involvement of G-protein(s). The effect of GTP[S] appeared to be specific, as up to 100 microM adenosine 5'-[gamma-thio]-triphosphate failed to stimulate PI-PLC activity, and guanosine 5'-[beta-thio]diphosphate inhibited activity. The response of membrane PI-PLC to Ca2+ was biphasic, while > 0.5 mM Mg2+ was inhibitory with or without GTP[S]. Comparable total PI-PLC activities and responses to GTP[S] and Ca2+ were observed in purified brush-border and basolateral membranes. Western immunoblots probed with monoclonal antibodies to PLC isoenzymes PLC-beta 1, -gamma 1 and -delta 1 demonstrated that these antipodal plasma membranes contain predominantly the PLC-delta 1 isoform, with small amounts of PLC-gamma 1 present but no detectable PLC-beta 1. PLC-gamma 1 was the major isoform detected in cytosol.
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PMID:Characterization of phosphoinositide-specific phospholipase C in rat colonocyte membranes. 838 28

An isozyme of phosphoinositide-specific phospholipase C (PLC) was purified to near homogeneity from bovine cerebellum by a combination of several column chromatography procedures. Approximately 80 micrograms of pure enzyme were obtained from 4 kg of bovine cerebellum, with a final specific activity of 7.5 mumol/min/mg protein in the presence of 0.1% deoxycholate. The enzyme is specific for phosphatidylinositol and phosphatidylinositol 4,5-bisphosphate but does not hydrolyze phosphatidylcholine. The molecular weight of the enzyme determined by SDS-polyacrylamide gel electrophoresis is approximately 97,000. Polyclonal antibodies to previously characterized PLC isozymes, PLC-beta 1, -beta 2, -gamma 1, -gamma 2, and - delta 1, did not cross-react with the purified cerebellar enzyme. Moreover, polyclonal antibodies prepared against the cerebellar enzyme did not react with purified PLC-beta 1, -beta 2, -gamma 1, -gamma 2, or -delta 1. However, the cerebellar enzyme was recognized by two antibodies generated against peptide sequences common to mammalian PLC isozymes. Comparison of partial amino acid sequences of the purified cerebellar enzyme with the deduced amino acid sequence of each known PLC isozyme shows that the cerebellar enzyme is a novel PLC, which could be classified as a PLC-beta-type isozyme. Thus, we have designated this enzyme PLC-beta 4.
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PMID:Purification of a novel phospholipase C isozyme from bovine cerebellum. 838 72

Cotransfection assays were used to show that the members of the GTP-binding protein Gq class of alpha subunits could activate phospholipase C (PLC) beta 2. Similar experiments also demonstrated that G beta 1 gamma 1, G beta 1 gamma 5, and G beta 2 gamma 5 could activate the beta 2 isoform of PLC but not the beta 1 isoform, while G beta 2 gamma 1 did not activate PLC beta 2. To determine which portions of PLC beta 2 are required for activation by G beta gamma or G alpha, a number of PLC beta 2 deletion mutants and chimeras composed of various portions of PLC beta 1 and PLC beta 2 were prepared. We identified the N-terminal segment of PLC beta 2 with amino acid sequence extending to the end of the Y box as the region required for activation by G beta gamma and the C-terminal region as the segment containing amino acid sequences required for activation by G alpha. Furthermore, we found that coexpression of G alpha 16 and G beta 1 gamma 1 but not G beta 1 gamma 5 in COS-7 cells was able to synergistically activate recombinant PLC beta 2. We suggest that G alpha 16 may act together with free G beta 1 gamma 1 to activate PLC beta 2, while G alpha 16 may form heterotrimeric complexes with G beta 1 gamma 5 and be stabilized in an inactive form. We conclude that the regions of PLC beta 2 required for activation by G beta gamma and G alpha are physically separate and that the nature of the G beta subunit may play a role in determining the relative specificity of the G beta gamma complex for effector activation while the nature of the G gamma subunit isoform may be important for determining the affinity of the G beta gamma complex for specific G alpha proteins.
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PMID:Activation of phospholipase C beta 2 by the alpha and beta gamma subunits of trimeric GTP-binding protein. 838 80

Xenopus oocytes exhibit a receptor-evoked Cl- current that is mediated through the activation of phospholipase C (PLC) and release of intracellular Ca2+. The identity of PLC(s) mediating this effect is unknown. We have cloned cDNAs encoding a new form of PLC-beta from a Xenopus oocyte cDNA library. The Xenopus PLC-beta has substantial (33-64%) homology with mammalian beta 1, beta 2, beta 3, and beta 4 phospholipase C and is closest to PLC-beta 3, with 64% identity and 80% similarity. Injection of antisense oligonucleotides to a specific region of Xenopus PLC-beta results in degradation of its mRNA and significantly reduces Cl- currents evoked by both endogenous angiotensin receptors and expressed mammalian alpha 1b-adrenergic receptors and M1-muscarinic receptors as compared to responses in sense oligonucleotide-injected oocytes. Inhibition of the M1-muscarinic response by antisense oligonucleotides was nonadditive with pertussis toxin inhibition. PLC antisense oligonucleotide-injected oocytes show Cl- current responses to IP3 that are indistinguishable from sense oligonucleotide-injected oocytes. Since the receptor responses are pertussis toxin-sensitive, we conclude that we have isolated a new form of PLC-beta involved in the pertussis toxin-sensitive receptor stimulation of the Ca2+ activated Cl- current in Xenopus oocytes.
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PMID:Receptor-evoked Cl- current in Xenopus oocytes is mediated through a beta-type phospholipase C. Cloning of a new form of the enzyme. 839 90


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