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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 metabolism of phosphatidylinositol (PtdIns) was studied in a mink lung epithelial cell line and its subclones transformed by feline sarcoma viruses containing either the v-fms or v-fes oncogenes. The transformed cell lines had a higher rate of PtdIns turnover but did not have elevated levels of phosphorylated PtdIns species or PtdIns kinase activity. Significantly higher specific activities of a guanine nucleotide-activated PtdIns-4,5-diphosphate phospholipase C were detected in both transformed cell lines (F3CL7(v-fes), 55 pmol/min/mg of protein and G2M(v-fms), 18 pmol/min/mg of protein) as compared to the nontransformed parental cell line (CCL64, 2 pmol/min/mg of protein). The guanine nucleotide-stimulated phospholipase C activity was specific for PtdIns-4,5-diphosphate, and the water-soluble hydrolysis product was inositol 1,4,5-triphosphate. Both GTP and nonhydrolyzable GTP analogs activated the phospholipase C, whereas ATP was weakly effective and GDP was inactive. The phospholipase C activity was maximally active in the presence of 9 mM sodium cholate, had a sharp pH optimum of pH 6.5, and was not activated by calcium although hydrolysis was inhibited by high concentrations of EDTA. These data point to enhanced production of diacylglycerol and inositol 1,4,5-triphosphate second messengers in transformed cells due to the activation of guanine nucleotide-dependent PtdIns-4,5-diphosphate-specific phospholipase C and suggest that the generation of aberrant hormonally independent signals is associated with cell transformation by oncogenes encoding tyrosine-specific protein kinases.
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PMID:A guanine nucleotide-dependent phosphatidylinositol 4,5-diphosphate phospholipase C in cells transformed by the v-fms and v-fes oncogenes. 300 85

From various rat organs, alkaline phosphodiesterase I was liberated by the action of phosphatidylinositol-specific phospholipase C obtained from Bacillus thuringiensis. Especially, a large amount of alkaline phosphodiesterase I was released from slices of small intestine, testis, lung, and kidney, but not from pancreas and liver. The release of the enzyme induced by phospholipase C was dependent on, or proportional to, the reaction time and the concentrations of the phospholipase C and the weight of the slices of small intestine or testis. Furthermore, little enzyme was released from the homogenate of pancreas. These results suggest an important role of phosphatidylinositol in the binding of alkaline phosphodiesterase I to the plasma membranes of rat small intestine and pancreas. The alkaline phosphodiesterase I released from slices of rat small intestine and testis had a molecular weight of about 240,000, and was activated by Mg2+ and Ca2+ but inhibited by EDTA. The enzyme hydrolyzed the phosphodiester linkage of p-nitrophenyl-thymidine 5'-monophosphate at pH 8.9, having the Km values of 0.36 mM (small intestine) and 0.25 mM (testis). The intestinal enzyme differed from the testis enzyme in pI values, thermostability, and Arrhenius plot having a single breakpoint.
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PMID:Alkaline phosphodiesterase I release from eucaryotic plasma membranes by phosphatidylinositol-specific phospholipase C. I. The release from rat organs. 301 61

Human platelets stimulated by epinephrine undergo enhanced turnover of phosphatidylinositol 4,5-bisphosphate, accumulate inositol trisphosphate, diacylglycerol, and phosphatidic acid, and phosphorylate a 47-kDa protein. All of these phenomena indicate stimulation of phospholipase C. These responses are blocked completely by inhibitors of alpha 2-adrenergic receptors (yohimbine), cyclooxygenase (aspirin or indomethacin), phospholipase A [2-(p-amylcinnamoyl)amino-4-chlorobenzoic acid (ONO-RS-082)], Na+/H+ exchange [ethylisopropylamiloride (EIPA)], fibrinogen binding to glycoprotein IIb/IIIa (antibody A2A9), Ca2+/Mg+ binding (EDTA), or removal of fibrinogen. Epinephrine evokes (i) an increased turnover of ester-linked arachidonic acid in aspirin treated platelets that is inhibited by ONO-RS-082, EDTA, yohimbine, or the absence of fibrinogen and (ii) a rapid cytoplasmic alkalinization that is inhibited partially by blockage of cyclooxygenase activity and completely by A2A9 or EIPA. In contrast, when incubated with subaggregatory concentrations of the prostaglandin H2/thromboxane A2 analogue [(15S)-hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic acid (U46619) and epinephrine, aspirin-treated platelets show a potentiation of phospholipase C activation that is unaffected by the above inhibitors. We propose that epinephrine, in promoting exposure of glycoprotein IIb/IIIa sites for fibrinogen binding, leads to a cytoplasmic alkalinization, which, in conjunction with local shifts in Ca2+, promotes low-level activation of phospholipase A. The resulting free arachidonic acid is converted to cyclooxygenase products, which, potentiated by epinephrine, activate phospholipase C. This further amplifies the initial stimulatory response.
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PMID:Activation of phospholipases A and C in human platelets exposed to epinephrine: role of glycoproteins IIb/IIIa and dual role of epinephrine. 302 70

Ectoenzyme release from porcine intestinal brush border membranes by phosphatidylinositol-specific phospholipase C of Bacillus thuringiensis was studied. Alkaline phosphodiesterase I, alkaline phosphatase and 5'-nucleotidase were released from both slices and brush border membranes. The pattern of alkaline phosphodiesterase I release was the same as that of alkaline phosphatase. The release of alkaline phosphodiesterase I induced by phospholipase C was dependent on, or proportional to, the reaction time and the concentration of phospholipase C. The Arrhenius plot for phosphodiesterase I release showed a single break at 30 degrees C for brush border membranes. Only 40% of alkaline phosphodiesterase I present in the brush border membranes were solubilized by phosphatidylinositol-specific phospholipase C treatment. The data indicate the presence of two forms of phosphodiesterase I, which are different in their sensitivity to phospholipase C. The released alkaline phosphodiesterase I had a molecular weight of 240,000 and was activated by Mg2+ and Ca2+, but strongly inhibited by EDTA.
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PMID:Alkaline phosphodiesterase I release from eucaryotic plasma membranes by phosphatidylinositol-specific phospholipase C. II. The release from brush border membranes of porcine intestine. 302

Thrombin, nucleotides, and chelators elicited a phosphatidylinositol 4,5-bisphosphate (PtdIns-P2) phospholipase C activity that was associated with human platelet membranes. Both alpha- and gamma-thrombin enhanced phospholipase C activity, whereas active site-inhibited alpha-thrombin did not stimulate PtdIns-P2 hydrolysis. PtdIns-P2 phospholipase C was also activated by nucleoside triphosphates, citrate, EDTA, and NaF. Magnesium was an inhibitor of PtdIns-P2 hydrolysis stimulated by nucleotides and chelators. Only PtdIns-P2 was degraded by the phospholipase C activated by alpha-thrombin, nucleotides, and chelators. The soluble fraction phospholipase C activity was also stimulated at low protein concentrations by nucleotides; however, soluble fraction phospholipase C activity cleaved both PtdIns-P2 and phosphatidylinositol 4-phosphate and was inhibited by chelators, suggesting the presence of a different enzyme in this compartment. The pH optimum for the membrane-associated phospholipase C in the presence of alpha-thrombin or nucleotides was 6.0, and the PtdIns-P2 phospholipase C was inhibited by neomycin and high detergent concentrations. Guanine nucleotides did not synergistically activate phospholipase C in the presence of alpha-thrombin. The characteristics of the membrane-associated PtdIns-P2 phospholipase C suggest that this enzyme is involved in platelet activation by the low-affinity alpha- or gamma-thrombin-dependent pathway.
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PMID:Thrombin- and nucleotide-activated phosphatidylinositol 4,5-bisphosphate phospholipase C in human platelet membranes. 303 33

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.
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PMID:Purification and characterization of two immunologically distinct phosphoinositide-specific phospholipases C from bovine brain. 304 Jul 53

A phospholipase C which hydrolyzes [14C]phosphatidylcholine has been purified 1782-fold from 70% ammonium sulfate extract of bull seminal plasma. Purification steps included acid precipitation, chromatography on DEAE-Sephacel, concanavalin A, octyl-Sepharose 4B and Ultrogel AcA 34. The final step provided homogeneous phospholipase C as determined by polyacrylamide gel electrophoresis. The enzyme comprised two subunits, Mr 69,000 and Mr 55,000, respectively. The enzyme had an optimum at pH 7.2 and pI 5.0. EDTA, Cd2+, Pb2+, Ni2+, Fe2+, and Zn2+ inhibited phospholipase C activity. Km and Vmax on p-nitrophenyl phosphorylcholine and phosphatidylcholine substrates were 20 mM and 17 mumol/min/mg of the purified enzyme and 100 microM and 18 mumol/min/mg of the purified enzyme, respectively. The enzyme appeared to be localized in the acrosome as judged by the binding of anti-phospholipase C to the acrosome. This phospholipase C, unlike other known phospholipases (C), did not hydrolyze [1-14C]phosphatidylinositol. The testicular extract of the guinea pig contained inactive phospholipase C which was activated on incubation with acrosin and trypsin but not chymotrypsin.
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PMID:Isolation and properties of a phosphatidylcholine-specific phospholipase C from bull seminal plasma. 308 12

The pathways for degradation of phosphatidylinositol (PI) were investigated in sonicated suspensions prepared from confluent cultures of bovine pulmonary artery endothelial cells. The time courses of formation of 3H-labeled and 14C-labeled metabolites of phosphatidyl-[3H]inositol ([3H]Ins-PI) and 1-stearoyl-2-[14C] arachidonoyl-PI were determined at 37 degrees C and pH 7.5 in the presence of 2 mM EDTA with or without a 2 mM excess of Ca2+. The rates of formation of lysophosphatidyl-[3H]inositol ([3H]Ins-lyso-PI) and 1-lyso-2-[14C] arachidonoyl-PI were similar in the presence and absence of Ca2+, and the absolute amounts of the two radiolabeled lyso-PI products formed were nearly identical. This indicated that lyso-PI was formed by phospholipase A1, and phospholipase A2 was not measurable. In the presence of EDTA, [14C]arachidonic acid release from 1-stearoyl-2-[14C]arachidonoyl-PI paralleled release of glycerophospho-[3H]inositol ([3H]GPI) from [3H]Ins-PI. Formation of [3H]GPI was inhibited by treatment with the specific sulfhydryl reagent, 2,2'-dithiodipyridine, and this was accompanied by an increase in [3H]Ins-lyso-PI. In the presence of Ca2+, [14C] arachidonic acid release from 1-stearoyl-2-[14C]arachidonoyl-PI was increased 2-fold and was associated with Ca2+-dependent phospholipase C activity. Under these conditions, [3H]inositol monophosphate production exceeded formation of [14C]arachidonic acid-labeled phospholipase C products, diacylglycerol plus monoacylglycerol, by an amount that was equal to the amount of [14C]arachidonic acid formed in excess of [3H]GPI. Low concentrations of phenylmethanesulfonyl fluoride (15-125 microM) inhibited Ca2+-dependent [14C]arachidonic acid release, and the decrease in [14C] arachidonic acid formed was matched by an equivalent increase in 14C label in diacylglycerol plus monoacyclglycerol. These data supported the existence of two pathways for arachidonic acid release from PI in endothelial cells; a phospholipase A1-lysophospholipase pathway that was Ca2+-independent and a phospholipase C-diacylglycerol lipase pathway that was Ca2+-dependent. The mean percentage of arachidonic acid released from PI via the phospholipase C-diacylglycerol lipase pathway in the presence of Ca2+ was 65 +/- 8%. The mean percentage of nonpolar phospholipase C products of PI metabolized via the diacylglycerol lipase pathway to free arachidonic acid was 28 +/- 3%.
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PMID:Ca2+-dependent and Ca2+-independent pathways for release of arachidonic acid from phosphatidylinositol in endothelial cells. 311 76

The role of G proteins in mediating adrenoceptor-prostacyclin synthesis coupling was investigated using the G protein activator, sodium fluoride. Sodium fluoride (NaF) stimulated in vitro rat aortic prostacyclin (PGI2) synthesis (EC50 = 5 x 10(-3) mol.l-1), an action inhibited completely by the presence of EDTA (10(-2) mol.l-1). The NaF-PGI2 dose-response curve was moved to the left by the presence of adrenaline, phorbol 12,13-dibutyrate (PDBU) and the Ca2+ ionophore A23187 in the incubation media. NaF-stimulated (5 x 10(-3) mol.l-1) PGI2 synthesis was inhibited by the Ca2+ channel blockers, verapamil and nifedipine, the protein kinase C inhibitor, H7, and lanthanum. Prazosin and yohimbine were without effect on NaF action, but partially inhibited adrenaline-potentiated NaF-stimulated PGI2 synthesis. Cyclic adenosine-3',5'-monophosphate (cAMP) and dibutyryl cAMP were without effect on de novo or NaF-, adrenaline-, PDBU- or A23187-stimulated PGI2 synthesis. Since fluoride is known to stimulate adenyl cyclase and phospholipase C, these data suggest that: (1) NaF stimulates in vitro rat aortic PGI2 synthesis by initiating Ca2+ influx; (2) this Ca2+ influx is mediated by protein kinase C, probably through G protein activation of phospholipase C and the generation of the protein kinase C activator, diacyl glycerol; and (3) adenyl cyclase and protein kinase A are not involved in NaF-stimulated PGI2 synthesis by the rat aorta.
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PMID:Fluoride stimulates in vitro vascular prostacyclin synthesis: interrelationship of G proteins and protein kinase C. 313 Nov 47

Staphylococcal alpha-toxin, a channel-forming protein, stimulates leukotriene B4 formation in rabbit polymorphonuclear leukocytes (PMN) (N. Suttorp, W. Seeger, J. Zucker-Reimann, L. Roka, and S. Bhakdi, Infect. Immun. 55:104-110, 1987). The concept was advanced that transmembrane toxin pores act as Ca2+ gates allowing passive Ca2+ influx into the cell, thus initiating stimulus response coupling. A critical step in this hypothesis is the demonstration of an increase in the cytosolic free Ca2+ concentration [( Ca2+]i). [Ca2+]i and membrane-associated Ca2+ were therefore monitored in quin-2- or chlorotetracycline-loaded PMN exposed to alpha-toxin. The effects of the Ca2+ ionophore ionomycin and the chemotactic tripeptide formylmethionyl-leucylphenylalanine (fMLP) were studied in parallel. All stimuli increased [Ca2+]i in dose- and time-dependent manner. In the presence of an EDTA excess there was a decrease of [Ca2+]i due to an efflux of Ca2+ in alpha-toxin- and ionomycin-treated cells, while addition of fMLP still induced an increase of [Ca2+]i. In the presence of verapamil, a Ca2+ channel blocker, [Ca2+]i was reduced after stimulation with fMLP but not with alpha-toxin or ionomycin. Addition of fMLP and ionomycin but not of alpha-toxin to PMN resulted in a rapid and substantial mobilization of membrane-associated Ca2+. The collective data demonstrate that exposure of PMN to staphylococcal alpha-toxin results in an increase in [Ca2+]i which is due to an influx of extracellular Ca2+ and not to a mobilization of intracellularly stored Ca2+. The concept of initiating stimulus response coupling by Ca2+ influx through transmembrane pores may be generally applicable to other channel-forming cytolysins.
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PMID:Effect of staphylococcal alpha-toxin on intracellular Ca2+ in polymorphonuclear leukocytes. 313 63


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