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)

The ability of bovine corpus luteum plasma membranes to bind 125I-choriogonadotropin has been examined after prior treatment of the membranes with phospholipases A, C, and D. Treatment of the purified membranes with low concentrations of phospholipases A and C resulted in the inhibition of the binding of 125I-choriogonadotropin to its receptors, whereas phospholipase D had no effect. Receptor activity was decreased by low concentrations of phospholipase A from either bee venom, Vipera russelli or Crotalus terrificus terrificus. Similarly, low concentrations of phospholipase C from Clostridium perfringens and Clostridium welchii also inhibited the binding activity while comparatively higher concentrations of phospholipase C from Bacillus cereus were required to achieve comparable inhibition. The time required to produce 50% inhibition of in vitro binding by phospholipases A and C was found to be 6 and 23 min, respectively. Upon either removal or chelation of calcium ions by ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid (EGTA) both enzymes were completely inhibited as evidenced by the complete retention of the membrane binding activity. The decrease in the specific binding of choriogonadotropin to membranes after phospholipase digestion resulted in a decrease in the number of binding sites and was not accompanied by a change in the affinity of the hormone-receptor complex. The rates of association and dissociation of the 125I-choriogonadotropin-receptor complex and the equilibrium dissociation constant (Kd) were nearly identical in untreated and phospholipase-treated membranes. Phospholipases did not have any effect on the preformed hormone-receptor complex or on solubilized receptor. Filtration through Sepharose 6B of solubilized 125I-choriogonadotropin-receptor complex from untreated membranes or membranes which had been pretreated with phospholipase C prior to carrying out hormone binding did not alter the profile (Kav 0.38). Gel filtration of membranes treated with phospholipase A showed two peaks of bound radioactivity with distribution coefficients (Kav) of 0.08 and 0.35, respectively.
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PMID:Gonadotropin receptors in plasma membranes of bovine corpus luteum. I. Effect of phospholipases on the binding of 125I-choriogonadotropin by membrane-associated and solubilized receptors. 18 85

The effects of several kinds of carbohydrate oxidase, SH-inhibitors and some other chemical reagents on the activities of von Willebrand factor, factor VIII procoagulant and factor VIII-related antigen were studied. Factor VIII procoagulant and von Willebrand factor activities were both inhibited by galactose oxidase, p-hydroxymercuribenzoic acid, 2,4,6-trinitrobenzensulfonic acid and sodium periodate, alpha-Mannosidase, N-ethylmaleimide and phospholipase C inactivated factor VIII procoagulant but not von Willebrand factor activity. Dithiothreitol had little effect on factor VIII procoagulant activity but reduced significantly that of von Willebrand factor. It is suggested that galactose and the thiol and epsilon-aminogroup groups of lysine may play an important role in both factor VIII procoagulant and von Willebrand factor activity. Mannose may be responsible for the factor VIII procoagulant activity but not for the von Willebrand factor activity. The Laurell rocket heights of factor VIII-related antigen rose with increasing concentration of galactose oxidase, 2,4,6-trinitrobenzenesulfonic acid or sodium periodate. Gel filtration experiments showed that factor VIII-related antigen may be dissociated into subunits by galactose oxidase but not by 2,4,6-trinitrobenzenesulfonic acid or sodium periodate.
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PMID:Studies of von Willebrand factor: effects of different kinds of carbohydrate oxidases, SH-inhibitors and some other chemical reagents. 30 2

Heat-stable enterotoxin (ST) produced by porcine strains of enterotoxigenic (ENT+) Escherichia coli has been purified to apparent homogeneity by sequential ultrafiltration, acetone fractionation, preparative gel electrophoresis, diethylaminoethyl Bio-Gel A ion-exchange chromatography, and Bio-Gel P-10 gel filtration. The enterotoxin, purified more than 1,500-fold, exhibited a molecular weight of 4,400, as determined by both sodium dodecyl sulfate-gel electrophoresis and gel filtration. A molecular weight of 5,100, representing 47 residues, was calculated from amino acid analysis data. The amino acid content was distinctive, with an unusually high proportion of cystines and few hydrophobic amino acids. A single amino-terminal residue, glycine, was observed. Purified ST was stable to heating (100 degrees C, 30 min) and did not lose biological activity after treatment with Pronase, trypsin, proteinase K, deoxyribonuclease, ribonuclease, and phospholipase C. Periodic acid oxidation and several organic solvents (acetone, phenol, chloroform, and methanol) had no effect on the biological activity of ST. Further, purified ST was stable to acid treatment at pH 1.0 but lost biological activity at pH values greater than 9.0. Neither lipopolysaccharide nor lipid contamination was evident in purified preparations. A characteristic absorption spectrum was observed during the course of the purification, which shifted from a maximum at 260 nm in crude preparations to 270 nm for the purified toxin. Antiserum obtained from rabbits immunized with ST or ST coupled to bovine serum albumin neutralized the action of the enterotoxin in suckling mice; however, passive hemagglutination and hemolysis titer assays suggested that ST is a poor antigen.
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PMID:Purification and chemical characterization of the heat-stable enterotoxin produced by porcine strains of enterotoxigenic Escherichia coli. 34 81

1. Lysosomes from rat liver contain two enzymic systems for hydrolysing phosphatidyl-inositol: a deacylation via lysophosphatidylinositol producing glycerophosphoinositol and non-esterified fatty acid, and a phospholipase C-like cleavage into inositol 1-phosphate and diaclygycerol. 2. The separate enzyme systems involved can be distinguished by gel filtration, differential temperature-stability and the inhibitory action of detergents. 3. The enzyme systems both have pH optima at 4.8 and their attack on a pure phosphatidylinositol substrate is inhibited by many bivalent metals including Ca2+ and Mg2+, and cationic drugs. 4. Whereas the deacylation system will attack other glycerophospholipids, the phospholipase C shows a marked specificity towards phosphatidylinositol, although it will also slowly attach phosphatidylcholine with the liberation of phosphocholine. 5. Gel filtration and temperature-stability distinguish the phospholipase C from lysosomal phosphatidic acid phosphatase, but not from sphingomyelinase. 6. Evidence is presented that an EDTA-insensitive phospholipase C degrading phosphatidylinositol is present in rat brain.
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PMID:The hydrolysis of phosphatidylinositol by lysosomal enzymes of rat liver and brain. 74 53

Gel filtration studies in the presence of Triton X-100 showed that treatment with phosphatidylinositol-specific phospholipase C reduced the apparent molecular size of the 100 kDa folate binding protein from human milk, choroid plexus and semen to 25 kDa. Cleavage of a hydrophobic glycosyl phosphatidylinositol domain (a membrane anchor) inserting the protein into Triton X-100 micelles could account for this phenomenon.
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PMID:Conversion of an apparent 100 kDa folate binding protein from human milk, choroid plexus and semen to a 25 kDa molecular species by phosphatidylinositol-specific phospholipase C. 133 54

Phosphoinositide specific phospholipase C from rabbit fast skeletal muscle has been enriched ca. 1,000-fold with a specific activity of 40 mumol x min-1 x mg-1. Following SDS-PAGE, renaturation of the enzyme protein in the presence of deoxycholate allowed the determination of an apparent molecular weight of 110 kDa. Gel-filtration of the native enzyme resulted in a very similar apparent molecular weight of 115 kDa, however, associated proteins of higher molecular weight were also found. Free Ca2+ concentrations needed for half-maximal activation of PtdIns(4,5)P2, PtdIns4P and PtdIns hydrolysis are 6.3 microM, 85 microM and 1.8 mM, and the Km values for these substrates 102, 340 and 937 microM, respectively.
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PMID:Rabbit fast skeletal muscle phospholipase C. Molecular weight determination by renaturation after polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate. 133 Jun 96

Thy-1 is abundantly expressed in the vertebrate nervous system. Perturbation studies in vitro suggest that Thy-1 inhibits neurite outgrowth and stabilizes neuronal processes (N. K. Mahanthappa and P. H. Patterson. (1992). Thy-1 involvement in neurite outgrowth: Perturbation by antibodies, phospholipase C, and mutation. Dev. Biol. 150,47-59). We here report that Thy-1 participates in several types of homophilic interactions, each with differential sensitivity to reduction and boiling. The relative abundance of the multimeric forms of Thy-1 vary with the cell's ability to sprout neurites. Gel filtration chromatography of sympathetic neuron and PC12 cell lysates reveals that Thy-1 immunoreactivity appears in 25-, 45-, and 150-kDa forms. In neurons, Thy-1 immunoreactivity is distributed equally in all three forms, whereas in PC12 cells, the majority of Thy-1 immunoreactivity is found in the higher molecular weight forms. When PC12 cells are induced to sprout neurites with NGF, the Thy-1 size distribution becomes identical to that of neurons. The three forms of Thy-1 immunoreactivity are likely to be homomultimers of Thy-1 because immunoaffinity-purified, soluble Thy-1 also forms complexes similar in size to those found in neuronal extracts. To test whether Thy-1 multimerization may occur through interactions like those between immunoglobulin heavy and light chains, synthetic peptides corresponding to candidate sites for such associations in Thy-1 were tested for their effects on multimerization and neurite outgrowth. One peptide increases the amount of monomeric Thy-1 relative to total Thy-1, and promotes outgrowth. These results suggest that multimeric forms of Thy-1 inhibit process outgrowth and neurite sprouting by stabilizing the surface membrane and/or underlying cytoskeleton.
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PMID:Thy-1 multimerization is correlated with neurite outgrowth. 134 22

We have purified to homogeneity the 33-kDa phosphatidylinositol-specific phospholipase C (PI-PLC) from the culture fluid of Listeria monocytogenes, a facultative intracellular pathogen. The protein was overexpressed, and secretion of PI-PLC was further enhanced by the addition of divalent cations to the culture medium. The basic protein (pI, approximately 9.4) was complexed with anionic proteins in the crude culture fluid. It bound to DEAE-Sepharose and was eluted from Sephacryl S-200 near the void volume in low-ionic-strength buffer, suggesting aggregates of greater than or equal to 150 kDa. Gel filtration chromatography on Sephacryl S-200 in the presence of 1 M ammonium sulfate resulted in disaggregation and complete separation of PI-PLC, which interacted with the column matrix. Amino-terminal sequencing of the pure protein gave results consistent with the previously deduced sequence and showed that the signal cleavage site was between alanine 29 and tyrosine 30. The enzyme was specific for PI and showed no activity with phosphatidylethanolamine, phosphatidylcholine, or phosphatidylserine. It did not cleave PI-4-phosphate or PI-4,5-bisphosphate, but it was active on the membrane form of the variable surface glycoprotein from Trypanosoma brucei, a PI-glycan-anchored protein. When assayed with deoxycholate-mixed micelles of PI, activity was highly dependent on added salt. Activation by salt was also observed with Triton X-100-mixed micelles. The optimal concentration of CaCl2 or MgCl2 was lower than that of KCl or (NH4)2SO4, but activity was not specifically dependent on divalent cations and was not inhibited by addition of EDTA. With deoxycholate, the optimum pH was 7.0. A broader pH optimum ranging from 5.5 to 6.5 was observed with Triton X-100-mixed micelles. These results are consistent with a postulated role for secreted PI-PLC in the acidified primary phagocytic vesicle of infected cells.
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PMID:Purification and characterization of Listeria monocytogenes phosphatidylinositol-specific phospholipase C. 139 18

The peptide angiotensin II (AngII) has been reported to stimulate phosphoinositide-specific phospholipase C (PLC) activity in the murine neuroblastoma cell line N1E-115. In the present study, polyclonal antibodies raised against a PLC isoenzyme, PLC-alpha, reacted with a 60-kDa protein present in both membrane and cytosolic fractions of differentiated N1E-115 cells. In order to examine the possible association of PLC-alpha with cell surface AngII receptors (AngII-Rs), membranes from differentiated N1E-115 cells were solubilized, using the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS). CHAPS (1%) solubilized AngII-Rs, from N1E-115 cells, that maintained their high affinity for agonists. Gel filtration analysis of the solubilized membranes revealed that the majority of the specific binding of 125I-AngII eluted as a large protein complex with a molecular mass of 380 kDa and that agonist binding was partially reduced by guanosine-5'-O-(3-thio)triphosphate (GTP gamma S), within this complex. CHAPS also effectively solubilized immunoreactive PLC-alpha, from N1E-115 cell membranes, that was similarly present within the 380-kDa AngII-binding complex. Anti-PLC-alpha antisera immunoprecipitated approximately 16% of the total phosphatidylinositol-4,5-bisphosphate-specific PLC activity in the 1% CHAPS extract and 40% of cytosolic PLC activity. Moreover, a 60-kDa 35S-Trans S-labeled protein, comigrating with immunoreactive PLC-alpha, was immunoprecipitated from the 1% CHAPS extract by the antisera. In addition, anti-PLC-alpha antisera immunoprecipitated approximately 20% of solubilized AngII-Rs prebound with 125I-AngII but failed to precipitate receptors prebound with the antagonist 125I-Sarc1,Ile8-AngII. The anti-PLC-alpha antisera also immunoprecipitated AngII-Rs when intact membranes were labeled with 125I-AngII before solubilization in 1% CHAPS, suggesting that the AngII-R interaction with PLC-alpha was not the result of detergent-promoted protein-protein interaction. On the other hand, monoclonal antibodies against another PLC isozyme, PLC-gamma, did not precipitate AngII-Rs in solubilized N1E-115 membranes. Finally, the formation of the immunoprecipitated AngII-R-PLC-alpha complex was disrupted by the nonhydrolyzable guanine nucleotide analog GTP gamma S, suggesting that the interaction between AngII-Rs and PLC-alpha is likely to involve a heterotrimeric guanine nucleotide-binding protein in neuron-like cells.
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PMID:Association of solubilized angiotensin II receptors with phospholipase C-alpha in murine neuroblastoma NIE-115 cells. 151 21

Plasma membranes from bovine liver contain a phosphatidylinositol 4,5-bisphosphate-specific phospholipase C (PLC) activity that is activated by guanine nucleotides. The G-proteins involved retained their ability to activate bovine brain PLC-beta 1 in a guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S)-dependent manner following extraction from the membranes with cholate and reconstitution with phospholipids. This reconstitution assay was used to purify the G-proteins by chromatography on heparin-Sepharose, DEAE-Sephacel, octyl-Sepharose, hydroxylapatite, Mono Q, and Sephacryl S-300 gel filtration. Gel electrophoresis showed that two alpha-subunits with molecular mass of 42 and 43 kDa were isolated to a high degree of purity, together with a beta-subunit. Neither alpha-subunit was a substrate for pertussis toxin-catalyzed ADP-ribosylation. Gel filtration of the final activity indicated an apparent molecular mass of 95 kDa, suggesting the presence of an alpha beta gamma heterotrimer. Immunological data revealed that the 42- and 43-kDa proteins were related to alpha-subunits of the Gq class recently purified from brain (Pang, I.-H., and Sternweis, P. C. (1990) J. Biol. Chem. 265, 18707-18712) and identified by molecular cloning (Strathmann, M., and Simon, M. I. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 9113-9117). The activation of PLC-beta 1 by the purified G-protein preparation was specific for nonhydrolyzable guanine nucleotides, the efficacy decreasing in order GTP gamma S greater than guanylimidodiphosphate greater than guanylyl(beta,gamma-methylene)-diphosphonate. Half-maximal activation required 4 microM GTP gamma S suggesting that the affinity of the G-proteins for GTP analogues is low. The GTP gamma S-dependent activation of PLC-beta 1 required millimolar Mg2+ and was inhibited by guanosine 5'-O-(2-thiodiphosphate) and by excess beta gamma-subunits. Aluminum fluoride also activated PLC-beta 1 in the presence of the G-proteins. The G-proteins were inactive toward PLC-gamma 1 or PLC-delta 1. In summary, these findings identify two G-protein activators of PLC-beta 1 that have the properties of heterotrimeric G-proteins and are members of the Gq class.
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PMID:Purification and characterization of two G-proteins that activate the beta 1 isozyme of phosphoinositide-specific phospholipase C. Identification as members of the Gq class. 165 41


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