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)

Phosphatidylinositol-specific phospholipase C (PI-PLC) was characterized in rat myocardium, and the effect of hypoxia on its activity was investigated. It had a substrate specificity toward phosphatidylinositol (PI) and was predominantly located in cytosol. Its optimal pH was 7.4 and it required 5 mM of Ca2+ for maximum activity, but did not hydrolyze phosphatidylcholine (PC), phosphatidylethanolamine (PE), or phosphatidylserine (PS). Vmax and Km were 51.5 nmol/mg/min, and 231 microM, respectively. Sodium deoxycholate increased its activity at a concentration of 0.05%, while Triton X-100 inhibited its activity at any concentrations examined. PI-PLC was partially purified 260 fold over the crude cytosol, with ammonium sulphate fractionation, DEAE-cellulose, Sephadex G-100, Hydroxylapatite, and Sephadex G-150 column chromatographies. In order to elucidate the biochemical function of myocardial PI-PLC in hypoxia, PI-PLC along with phospholipase A2 (PLA2) was investigated in N2 gas-saturated buffer up to for 24 hours. The activity of PI-PLC did not change during the first 2 hours, and then gradually attenuated. Substrate specificity or subcellular localization of PI-PLC unchanged during 24 hour 9 of hypoxia. PLA2 was predominantly located in microsome and had a substrate specificity toward PE in normoxic state. In hypoxia, on the other hand, it hydrolyzed PC besides PE and was activated on and after 2 hours of hypoxic incubation. PI-PLC did not seem to contribute in releasing arachidonate from membrane lipid-bilayers during myocardial ischemia. But some biochemical mechanism suggested to inhibit its activity protecting the abrupt cell damage.
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PMID:[Phosphatidylinositol-specific phospholipase C in hypoxic rat myocardium]. 282 21

Protein kinase C activity towards exogenous histone was found in a cytosolic fraction of rat renal mesangial cells. The analysis of the 100,000 x g supernatant fraction with DEAE-cellulose ion-exchange chromatography gave a protein kinase C preparation that was dependent on Ca2+ and phosphatidylserine for its activity. The addition of diolein decreased the Ca2+ requirement of the enzyme. 1-(5-Isoquinoline-sulfonyl)-2-methylpiperazine (H-7), sphingosine and cytotoxin I potently inhibited the protein kinase C activity prepared from mesangial cells as well as the 12-O-tetradecanoylphorbol 13-acetate (TPA)-induced prostaglandin synthesis in intact mesangial cells. In the second part of the study, the desensitization of angiotensin II-stimulated phospholipase C activity was investigated. Angiotensin II induced a rapid increase in inositol trisphosphate (IP3) formation. Pretreatment of cells with angiotensin II, followed by removal of the hormone, resulted in a decreased response to a second application of angiotensin II. A similar protocol involving pretreatment with angiotensin II had no effect on subsequent responsiveness to [Arg8]vasopressin. The specific antagonist [Sar1, Ala8]angiotensin II did not stimulate IP3 formation neither did it inhibit the response to a subsequent stimulation with angiotensin II. After angiotensin II pretreatment, a prolonged incubation (120 min) restored responsiveness of the cells to angiotensin II. Pretreatment of mesangial cells with H-7, sphingosine or cytotoxin I almost completely diminished the desensitization of angiotensin II-stimulated IP3 generation. These results indicate that, in rat mesangial cells, angiotensin II induces a homologous desensitization of phospholipase C stimulation. It is proposed that protein kinase C activation plays an important role in the molecular mechanism of desensitization of angiotensin II-stimulated polyphosphoinositide metabolism.
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PMID:Protein kinase C from rat renal mesangial cells: its role in homologous desensitization of angiotensin II-induced polyphosphoinositide hydrolysis. 283 88

A soluble phospholipase C from rat liver was purified to homogeneity using phosphatidylinositol 4,5-bisphosphate (PIP2) as substrate. After ammonium sulfate fractionation, the purification involved chromatography on phosphocellulose, DEAE-Sepharose CL-6B, hydroxylapatite, Reactive Blue 2 dye-linked agarose, and Mono S cation exchanger. Under the conditions of the assay, the pure enzyme had a specific activity of 407 mumol/mg protein/min. It migrated as a single band with a molecular mass of 87 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The water-soluble product formed during the hydrolysis of PIP2 by the purified enzyme was inositol 1,4,5-trisphosphate. The enzyme shows one-half of maximum velocity at 2 microM Ca2+ with PIP2 as substrate. Between 0 and 100 microM Ca2+, the enzyme shows approximately the same activity with phosphatidylinositol 4-phosphate (PIP) as it does with PIP2, and very low activity with phosphatidylinositol. The enzyme is activated by low concentrations of basic proteins; for example, with PIP2 as substrate, 1 microgram/ml histone activates the enzyme 3.6-fold. The enzyme shows an almost absolute requirement for monovalent salts which can be met by different alkali metal halides. A second, minor peak of PIP2-hydrolyzing phospholipase C activity was resolved during chromatography of the enzyme on hydroxylapatite. The substrate specificity suggests that PIP and PIP2 are normal substrates of this enzyme. Under physiological conditions of activation, the enzyme may therefore generate inositol 1,4-bisphosphate and inositol 1,4,5-trisphosphate in amounts determined by the ratio of PIP and PIP2 present in the cellular membranes.
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PMID:Purification of a phospholipase C from rat liver cytosol that acts on phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 4-phosphate. 284 77

Two forms of phospholipase C, hydrolyzing specifically inositol phospholipids, are resoluted and partially purified from rat brain as well as liver cytosol by DEAE-cellulose followed by heparin-Sepharose, Sephacryl S-400, and aminohexyl-Sepharose column chromatographies. With phosphatidylinositol as substrate, at pH 7.4 one is most active at 10(-6) M Ca2+ (Type I) whereas the other requires 10(-3) M Ca2+ (Type II). At pH 5.5 both Type I and II are active at 10(-3) M Ca2+ but essentially inactive at lower concentrations of this divalent cation. Both Type I and II hydrolyze preferentially polyphosphoinositides particularly at lower concentrations of Ca2+.
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PMID:Rat brain and liver soluble phospholipase C: resolution of two forms with different requirements for calcium. 299 75

Three forms (I, IIa and IIb) of phospholipase C, hydrolyzing specifically inositol phospholipids, were resolved from human platelet cytosol and partially purified by DEAE-cellulose, phenyl-Sepharose, Ultrogel ACA-44 and hydroxylapatite column chromatographies. All three forms exhibited pH optimum at 6.5 - 7.0 in the presence of deoxycholate and their molecular weights were 67,000 (form I), 120,000 (IIa) and 70,000 (IIb). These enzymes hydrolyzed both phosphatidylinositol and phosphatidylinositol 4,5-bisphosphate in Ca2+-dependent manner; their maximal activities for phosphatidylinositol hydrolysis were obtained at 10(-4) to 10(-3) M Ca2+, whereas phosphatidylinositol 4,5-bisphosphate was preferentially hydrolyzed at lower concentration of Ca2+.
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PMID:Partial purification of phosphoinositide phospholipase C from human platelet cytosol; characterization of its three forms. 301 Sep 93

Insulin stimulates the hydrolysis of a phosphatidylinositol-glycan, resulting in the generation of two related inositol phosphate-glycan enzyme modulators and diacylglycerol. A phosphatidylinositol-glycan-specific phospholipase C that catalyzed this reaction was found in the plasma-membrane fraction of rat liver. The enzymatic activity was measured by the release of diacylglycerol from the glycosylated-phosphatidylinositol-membrane-anchored variant surface glycoproteins of African trypanosomes. The enzyme also catalyzed the production of an inositol phosphate-glycan from an insulin-sensitive phosphatidylinositol-glycan precursor. The enzyme was solubilized with neutral nonionic detergent and purified to near homogeneity by anion-exchange chromatography on DEAE-cellulose, followed by hydrophobic chromatography on butyl-agarose. The resulting enzyme preparation was purified approximately 20,000-fold from whole liver and exhibited one major silver-stained band of Mr 52,000 on NaDodSO4/PAGE. Gel permeation chromatography of the purified activity revealed a Stokes radius of 35 A and an apparent molecular weight of 62,000, suggesting that the enzyme was tightly associated with lipid or detergent but existed as a monomer in its active form. The enzyme was specific for glycosylated phosphatidylinositol; no hydrolysis of phosphatidylinositol, phosphatidylinositol 4,5-bisphosphate, or phosphatidylcholine was observed. The enzyme was calcium independent and thiol activated. These data suggest a role for the phosphatidylinositol-glycan-specific phospholipase C as an effector for some of the metabolic actions of insulin.
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PMID:Purification of a phosphatidylinositol-glycan-specific phospholipase C from liver plasma membranes: a possible target of insulin action. 303 58

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

A soluble inositolphospholipid-specific phospholipase C (PI-phospholipase C) has been purified 5,800-fold from the cytosolic fraction of calf thymocytes. The purification was achieved by sequential column chromatographies on DEAE-Sepharose CL-6B, heparin-Sepharose CL-6B, Sephacryl S-300, Mono S, and Superose 12, followed by column chromatography on Sephadex G-100 in the presence of 1% sodium cholate. The enzyme thus purified was found to be homogeneous on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weight of the enzyme was estimated to be 68 kDa by SDS-PAGE. The enzyme is specific for inositol phospholipids. Phosphatidylinositol and phosphatidylinositol 4,5-bisphosphate (PIP2) were hydrolyzed, but phosphatidylcholine and phosphatidylethanolamine were not affected by the enzyme. GTP gamma S-binding activity was detected in the enzyme fractions after all the purification steps, but not in the final enzyme preparation. The PI-phospholipase C and GTP gamma S-binding activities in the partially purified enzyme preparation could be separated by the column chromatography on Sephadex G-100 only in the presence of 1% sodium cholate. Thus, the soluble PI-phospholipase C has affinity to a GTP-binding protein. SDS-PAGE of the GTP-binding fractions eluted from the Sephadex G-100 column gave three visible bands of 54, 41, and 27 kDa polypeptide was specifically ADP-ribosylated by pertussis toxin. Furthermore, it was found that GTP and GTP gamma S (10 microM and 1 mM) could enhance the PIP2 hydrolysis activity of the partially purified enzyme in the presence of 3 mM EGTA, but the purified enzyme after separation from the GTP-binding activity was not affected by GTP and GTP gamma S. The soluble PI-phospholipase C of calf thymocytes may be not only physically but also functionally associated with a GTP-binding protein.
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PMID:Physical and functional association of cytosolic inositolphospholipid-specific phospholipase C of calf thymocytes with a GTP-binding protein. 312 64

Recent studies have shown that the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA) stimulates protein kinase C (PKC), whereas the ether-linked phospholipid 1-O-octadecyl-2-O-methyl-rac-glycerol-3-phosphocholine (ET-18-OCH3) inhibits PKC activity in vitro. Therefore, the antitumor effects of ET-18-OCH3 could be due to its inhibition of PKC activity and the effects of tumor promotion. TPA stimulates arachidonic acid release, prostaglandin synthesis, phosphatidylcholine synthesis and the degradation of phosphatidylcholine by phospholipase C in Madin Darby canine kidney (MDCK) cells. Therefore, we have determined the effects of ET-18-OCH3 on these consequences of TPA stimulation. Preliminary experiments determined that ET-18-OCH3 inhibited PKC partially purified from MDCK cells by ion-exchange chromatography on DEAE-cellulose. In addition, ET-18-OCH3 inhibited the TPA-stimulated phosphorylation of a 40,000-dalton protein in intact MDCK cells. These data indicate that ET-18-OCH3 is an effective inhibitor of PKC activity in MDCK cells. In addition, ET-18-OCH3 was found to inhibit arachidonic acid release and prostaglandin synthesis. The inhibition of prostaglandin synthesis appears to be secondary to inhibition of arachidonic acid release, since ET-18-OCH3 does not inhibit TPA-stimulated synthesis of prostaglandin H synthase or the activity of the enzyme directly (Parker, J., Daniel, L. W., and Waite, M. [1987] J. Biol. Chem. 262, 5385-5393). ET-18-OCH3 also inhibits TPA-stimulated phosphatidylcholine synthesis and phosphatidylcholine degradation by phospholipase C.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Ether lipids inhibit the effects of phorbol diester tumor promoters. 344 77

An enzyme hydrolyzing sphingomyelin was purified from extracts of solid cultures of Aspergillus saitoi 7041 by fractionation with isopropanol followed by successive column chromatographies on DEAE-Sepharose CL-6B, butyl-Toyopearl 650 M, and phenyl-Sepharose CL-4B. The preparation of purified enzyme was homogeneous and had an activity increased 81-fold over that of the isopropanol fraction. The yield was about 65%. The molecular weight was estimated to be 54,000 by sodium dodecyl sulfate-gel electrophoresis. The enzyme solution had a violet color and contained iron atoms. The enzyme catalyzed the hydrolysis of sphingomyelin to N-acylsphingosine and phosphorylcholine. The optimum pH for hydrolytic activity was around 3.5. The Km values for sphingomyelin and 2-hexadecanoylamino-4-nitrophenylphosphorylcholine were 0.11 and 0.33 mM, respectively. The enzyme also catalyzed the hydrolysis of other phospholipids; the order of its hydrolytic activity at a substrate concentration of 2.5 mM was phosphatidylcholine greater than or equal to sphingomyelin = phosphatidylethanolamine = lysophosphatidylethanolamine greater than phosphatidyl DL-glycerol = phosphatidyl L-serine greater than phosphatidylinositol. From these results, this enzyme appears to be a new type of phospholipase C(phosphatidylcholine cholinephosphohydrolase, EC 3.1.4.3).
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PMID:Purification and properties of a phospholipase C that has high activity toward sphingomyelin from Aspergillus saitoi. 367 75


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