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)

Aminopeptidase P (EC 3.4.11.9) was solubilized from pig kidney membranes with bacterial phosphatidylinositol-specific phospholipase C (PI-PLC) and then purified by a combination of anion-exchange and hydrophobic-interaction chromatographies. Contaminating peptidase activities were removed by selective affinity chromatography. The purified enzyme was apparently homogeneous on SDS/PAGE with an Mr of 91,000. Enzymic deglycosylation revealed that aminopeptidase P is a glycoprotein, with up to 25% by weight of the protein being due to the presence of N-linked sugars. The phospholipase-solubilized aminopeptidase P was recognized by an antiserum to the cross-reacting determinant (CRD) characteristic of the glycosyl-phosphatidylinositol anchor. This recognition was abolished by mild acid treatment or deamination with HNO2, indicating that the CRD was due exclusively to the inositol 1,2-cyclic phosphate ring epitope generated by the action of PI-PLC. The activity of aminopeptidase P was inhibited by chelating agents and was stimulated by Mn2+ or Co2+ ions, confirming the metallo-enzyme nature of this peptidase. Selective inhibitors of other aminopeptidases (actinonin, amastatin, bestatin and puromycin) had little or no inhibitory effect.
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PMID:Purification and characterization of pig kidney aminopeptidase P. A glycosyl-phosphatidylinositol-anchored ectoenzyme. 213 78

The mechanisms of endothelin-1 (ET) actions were investigated in cultured rat aortic vascular smooth muscle A-10 cells. The A-10 cells have a single class of high affinity binding sites for ET with an apparent Mr of 65,000-75,000 on SDS-PAGE. Stimulation of cells with ET induces mobilization of Ca2+ from both intra- and extracellular pools to produce a biphasic increase in cytoplasmic free Ca2+ concentration. ET increases cellular levels of inositol trisphosphate and 1,2-diacylglycerol, indicating activation of phospholipase C by ET. ET stimulates production of inositol phosphates in membranes prepared from A-10 cells in the presence of guanosine 5'-O-(thiotriphosphate) (GTP gamma S), but not in its absence. Further, specific binding of 125I-labeled ET to A-10 cell membranes is shown to be inhibited by GTP gamma S in a dose-dependent manner. Treatment of A-10 cells with pertussis toxin induces ADP-ribosylation of a 41,000-D membrane protein but fails to block the ET-induced increases in inositol phosphate production and Ca2+ mobilization. These results indicate that the receptor for ET is coupled to phospholipase C via a guanine nucleotide-binding regulatory protein which is distinct from the pertussis toxin substrate in A-10 cells.
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PMID:Endothelin receptor is coupled to phospholipase C via a pertussis toxin-insensitive guanine nucleotide-binding regulatory protein in vascular smooth muscle cells. 215 22

A human platelet cytosolic phosphoinositide-specific phospholipase C, one of four PLC activity peaks separated by column chromatographies, designated as cPLC-I, was purified to homogeneity. The cPLC-I exhibited an apparent Mr of 145 kDa by SDS-polyacrylamide gel electrophoresis and was immunologically identified to be PLC-gamma 2. It hydrolyzed PI and PIP2 at optimum pH of 5.5-6.0. Deoxycholate and cholate inhibited the enzyme activity to hydrolyze two substrates. Calcium was required to obtain the maximal activity for PI- and PIP2-hydrolysis at concentration of 10(-3) M and 10(-5) M, respectively. Hg2+ (1 microM) inhibited strongly the enzyme activity.
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PMID:Purification and characterization of a cytosolic phosphoinositide-phospholipase C (gamma 2-type) from human platelets. 215 3

Basic carboxypeptidase activity was released from human placental membranes on treatment with phosphatidylinositol-specific phospholipase C of Bacillus thuringiensis. The enzyme was successively purified to homogeneity by SDS-polyacrylamide gel electrophoresis. The molecular nature and some catalytic properties of the purified enzyme revealed that it is identical with recently described basic carboxypeptidase M (R.A. Skidgel et al. J. Biol. Chem. 264 (4) 1989 2236-2241).
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PMID:Human placental carboxypeptidase M is anchored by a glycosyl-phosphatidylinositol moiety. 216 Dec 29

A novel bovine spleen phosphoinositide-specific phospholipase C (PLC) has been identified with respect to immunoreactivity with four independent antibodies against each of the PLC isoenzymes, and purified to near homogeneity by sequential column chromatography. Spleen contains three of the isoenzymes: two different gamma-types [gamma 1 and gamma 2, originally named as PLC-gamma [Rhee, Suh, Ryu & Lee (1989) Science 244, 546-550] and PLC-IV [Emori, Homma, Sorimachi, Kawasaki, Nakanishi, Suzuki & Takenawa (1989) J. Biol. Chem. 264, 21885-21890] respectively] and delta-type of the enzyme, but PLC-gamma 1 is separated from the PLC-gamma 2 pool by the first DEAE-cellulose column chromatography. Subsequently, PLC-delta is dissociated on the third heparin-Sepharose column chromatography. The purified enzyme has a molecular mass of 145 kDa on SDS/polyacrylamide-gel electrophoresis and a specific activity of 12.8 mumol/min per mg with phosphatidylinositol 4,5-bisphosphate as substrate. This enzyme activity is dependent on Ca2+ for hydrolysis of all these phosphoinositides. None of the other phospholipids examined could be its substrate at any concentration of Ca2+. The optimal pH of the enzyme is slightly acidic (pH 5.0-6.5).
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PMID:Isolation and characterization of a gamma-type phosphoinositide-specific phospholipase C (PLC-gamma 2). 216 90

Eighty-three percent of polyphosphoinositide-specific phospholipase C activity was recovered in a cytosolic fraction after nitrogen cavitation of turkey erythrocytes. This activity has been purified approximately 50,000-fold when compared to the starting cytosol with a yield of 1.7-5.0%. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the phospholipase C preparation revealed a major polypeptide of 150 kDa. The specific activity of the purified enzyme was 6.7-14.0 mumol/min/mg of protein with phosphatidylinositol 4,5-bisphosphate or phosphatidylinositol 4-phosphate as substrate. Phospholipase C activity was markedly dependent on the presence of Ca2+. The phospholipase C showed an acidic pH optimum (pH 4.0). At neutral pH, noncyclic inositol phosphates were the major products formed by the phospholipase C, while at pH 4.0, substantial formation of inositol 1:2-cyclic phosphate derivatives occurred. Properties of the purified 150-kDa turkey erythrocyte phospholipase C were compared with the approximately 150-kDa phospholipase C-beta and -gamma isoenzymes previously purified from bovine brain (Ryu, S. H., Cho, K. S., Lee, K. Y., Suh, P. G., and Rhee, S. G. (1987) J. Biol. Chem. 262, 12511-12518). The turkey erythrocyte phospholipase C differed from the two mammalian phospholipases with respect to the effect of sodium cholate on the rate of polyphosphoinositide hydrolysis observed. Moreover, when presented with dispersions of pure inositol lipids, phospholipases C-beta and -gamma displayed comparable maximal rates of polyphosphoinositide and phosphatidylinositol hydrolysis. By contrast, the turkey erythrocyte phospholipase C displays a marked preference for polyphosphoinositide substrates.
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PMID:A receptor and G-protein-regulated polyphosphoinositide-specific phospholipase C from turkey erythrocytes. I. Purification and properties. 216 32

Two isozymes of phosphoinositide-specific phospholipase C were isolated and purified from salt-washed rabbit brain membranes. The membranes were extensively washed with isotonic, hypertonic and hypotonic buffers prior to solubilization with sodium cholate. Two isozymes (PLC-IV and PLC-beta m) were purified by a combination of DEAE-Sephacel, AH-Sepharose, heparin-Sepharose, AcA-34 gel filtration and mono-Q FPLC chromatographies. The major activity (PLC-beta m) was purified to homogeneity and had an estimated molecular weight of 155,000 on sodium-dodecyl sulfate-polyacrylamide gels (SDS-PAGE). This isozyme was immunologically identified as PLC-beta, an isozyme previously characterized in bovine brain cytosol and 2 M KCl membrane extracts. A second isozyme, PLC-IV, was immunologically distinct from PLC-beta and PLC-gamma and was purified to a stage where three protein bands (Mr 66,000, 61,000 and 54,000) on SDS-PAGE correlated with enzyme activity. The catalytic properties of the isozymes were studied and found to be very similar. The specific activities for PIP2 were greater than those obtained when PI was used. Both PLC-IV and PLC-beta m were Ca2(+)-dependent; near maximal stimulation for PI and PIP2 hydrolysis was observed at 0.5 microM free Ca2+. Sodium pyrophosphate and sodium fluoride stimulated phospholipase C activity of both isozymes. Polyclonal antibodies raised against PLC-beta m were able to inhibit carbachol and GTP gamma S stimulated phospholipase C activity in 2 M KCl washed rabbit cortical membranes. This suggests that in rabbit brain muscarinic cholinergic stimulation regulates PLC-beta m.
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PMID:Purification and characterization of PLC-beta m, a muscarinic cholinergic regulated phospholipase C from rabbit brain membrane. 216 89

Schwann cells synthesize both hydrophobic and peripheral cell surface heparan sulfate proteoglycans (HSPGs). Previous analysis of the kinetics of radiolabeling suggested the peripheral HSPGs are derived from the membrane-anchored forms (Carey, D., and D. Evans. 1989. J. Cell Biol. 108:1891-1897). Peripheral cell surface HSPGs were purified from phytic acid extracts of cultured neonatal rat sciatic nerve Schwann cells by anion exchange, gel filtration, and laminin-affinity chromatography. Approximately 250 micrograms of HSPG protein was obtained from 2 X 10(9) cells with an estimated recovery of 23% and an overall purification of approximately 2000-fold. SDS-PAGE analysis indicated the absence of non-HSPG proteins in the purified material. Analysis of heparinase digestion products revealed the presence of at least six core protein species ranging in molecular weight from 57,000 to 185,000. The purified HSPGs were used to produce polyclonal antisera in rabbits. The antisera immunoprecipitated a subpopulation of 35SO4-labeled HSPGs that were released from Schwann cells by incubation in medium containing phosphatidylinositol-specific phospholipase C (PI-PLC); smaller amounts of immunoprecipated HSPGs were also present in phytic acid extracts. In the presence of excess unlabeled PI-PLC-released proteins, immunoprecipitation of phytic acid-solubilized HSPGs was inhibited. SDS-PAGE analysis of proteins immunoprecipitated from extracts of [35S]methionine labeled Schwann cells demonstrated that the antisera precipitated an HSPG species that was present in the pool of proteins released by PI-PLC, with smaller amounts present in phytic acid extracts. Nitrous acid degradation of the immunoprecipitated proteins produced a single 67,000-Mr core protein. When used for indirect immunofluorescence labeling, the antisera stained the external surface of cultured Schwann cells. Preincubation of the cultures in medium containing PI-PLC but not phytic acid significantly reduced the cell surface staining. The antisera stained the outer ring of Schwann cell membrane in sections of adult rat sciatic nerve but did not stain myelin or axonal membranes. This localization suggests the HSPG may play a role in binding the Schwann cell plasma membrane to the adjacent basement membrane surrounding the individual axon-Schwann cell units.
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PMID:Identification of a lipid-anchored heparan sulfate proteoglycan in Schwann cells. 217 60

Lipophosphoglycan was isolated from the dividing, noninfective stage and from the nondividing metacyclic stage of Leishmania major promastigotes. The lipophosphoglycans were characterized by SDS-PAGE and by chromatographic and quantitative analysis of phosphatidylinositol-specific phospholipase C- and mild acid-generated fragments. The results revealed two stage-specific structural differences: (i) an increase in size of the metacyclic form of the glycoconjugate due to an approximate doubling in the number of its salient phosphorylated saccharide units; and concomitantly, (ii) a subtle compositional change in these units. Gas-liquid chromatographic analysis indicated that the phosphatidylinositol lipid anchor was developmentally conserved. These developmental modifications suggest important roles for the lipophosphoglycan which have not been previously considered, such as promoting complement resistance within the vertebrate host, and midgut attachment and release within the sand fly vector.
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PMID:Developmental modification of the lipophosphoglycan from Leishmania major promastigotes during metacyclogenesis. 217 18

A postsynaptic acting short chain alpha-toxin, B.f. III, was isolated from venom of the banded krait (Bungarus fasciatus) using ion-exchange chromatography. The toxin, a basic protein (pI = 10) has an apparent molecular weight of 6,500 as determined by SDS-polyacrylamide gel electrophoresis. It shares immunological determinants with alpha-bungarotoxin, as it cross-reacted with antibodies raised in rabbits against alpha-bungarotoxin. B.f. III inhibits binding of 125I-alpha-bungarotoxin to cultured chick myotubes with an IC50 of 3 X 10(-10) M. The rate of association with chick myotube nAChR was 3 times faster than that of alpha-bungarotoxin, and binding was slowly reversible. The toxin is a less potent antagonist than alpha-bungarotoxin; in ion flux experiments, measuring influx of 86Rb in chick myotubes, B.f. III inhibited carbachol-induced influx of 86Rb (IC50 = 5 X 10(-9) M) at concentrations higher than those needed for alpha-bungarotoxin (IC50 = 6 X 10(-10) M).
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PMID:Interaction with chick myotube cholinergic receptors of an alpha-neurotoxin isolated from venom of the banded krait (Bungarus fasciatus). 243 Mar 47


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