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)

The Leu-8 molecule, the human homologue of the murine MEL-14 peripheral lymph node homing receptor, is expressed on neutrophils in both species and may be important in localization of cells to sites of inflammation. Most circulating human neutrophils express the Leu-8 molecule, and activation of neutrophils with phorbol myristate acetate causes a rapid decline in Leu-8 membrane fluorescence staining within 15 minutes. Northern blot analysis of total cellular RNA from neutrophils demonstrated two species of Leu-8 messenger RNA, a major one of 2.4 kb and a minor one of 1.9 kb. Because two different Leu-8 cDNA clones were obtained from human lymphocytes that were predicted to encode both transmembrane and phosphatidylinositol (PI)-anchored forms of the molecule, experiments were conducted to determine whether Leu-8 is anchored to neutrophils by a PI-anchor. There was a slight decrease in expression of Leu-8 on neutrophils when they were treated with PI-specific phospholipase C (PI-PLC). However, Leu-8 was abundant on neutrophils obtained from a patient with paroxysmal nocturnal hemoglobinuria. To determine the fate of the Leu-8 molecule during cell activation, neutrophils were labeled with 125I-anti-Leu-8. During activation antibody was rapidly lost from the cell surface and was not internalized, suggesting that Leu-8 is released from the cell membrane during cell activation. When cell extracts of neutrophils were compared with extracts of lymphoid cells by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting, the Leu-8 species expressed on neutrophils had a significantly higher and more variable relative mobility (70 to 120 Kd for neutrophils v 70 Kd for Jurkat T cells). In addition, Leu-8 molecules were detected in the supernatants of activated neutrophils. These results indicate that human neutrophils express a high-molecular-weight form of the Leu-8 molecule that has a conventional transmembrane anchor and is rapidly released from the membrane during activation. The loss of the Leu-8 membrane glycoprotein during activation may be a mechanism for rapid alteration of neutrophil adhesion characteristics.
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PMID:Human neutrophils release the Leu-8 lymph node homing receptor during cell activation. 170 70

Platelet-derived growth factor (PDGF) stimulates autophosphorylation of the PDGF receptor and association of the receptor with several cytoplasmic molecules, including phosphatidylinositol-3 kinase (PI3 kinase). In this study we examined the association of PI3 kinase with immunoprecipitated autophosphorylated PDGF receptor in vitro. The PI3 kinase from cell lysates bound to the wild-type receptor but not to a mutant receptor that had a deletion of the kinase insert region. A protein of an apparent size of 85 kDa bound to the receptor, consistent with previous observations that a protein of this size is associated with PI3 kinase activity. In addition, 110- and 74-kDa proteins bound to the phosphorylated receptor. Dephosphorylated receptors lost the ability to bind PI3 kinase activity as well as the 85-kDa protein. A 20-amino-acid peptide composed of a sequence in the kinase insert region that included one of the autophosphorylation sites of the receptor (tyrosine 719) as well as a nearby tyrosine (Y708) blocked the binding of PI3 kinase to the receptor, but only when the peptide was phosphorylated on tyrosine residues. A scrambled version of the peptide did not block PI3 kinase binding to the receptor even when it was phosphorylated on tyrosine. These tyrosine-phosphorylated peptides did not block binding of phospholipase C-gamma or GTPase-activating protein to the receptor. In separate experiments (receptor blots), soluble radiolabeled receptor bound specifically to an 85-kDa protein present in sodium dodecyl sulfate-polyacrylamide gel electrophoresis-fractionated 3T3 cell lysates that were transferred to nitrocellulose paper. The binding was blocked by the same tyrosine-phosphorylated peptides that prevented binding of PI3 kinase activity to immobilized receptors. These findings show that the PDGF receptor binds directly to an 85-kDa protein and to a PI3 kinase activity through specific sequences in the kinase insert region. The association of a 110-kDa protein with the receptor also involve these sequences, suggesting that this protein may be a subunit of the PI3 kinase. Phosphotyrosine is an essential structure required for the interactions of these proteins with the PDGF receptor.
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PMID:A phosphatidylinositol-3 kinase binds to platelet-derived growth factor receptors through a specific receptor sequence containing phosphotyrosine. 170 28

Monoclonal antibody BM88 recognizes a neurospecific surface antigen in the CNS and the PNS. In the present study, the antigen recognized by BM88 was immunopurified from pig brain and shown to be a 22-kDa polypeptide by reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Under nonreducing conditions a protein of 40 kDa was obtained, a result indicating that the antigen is composed of two polypeptide chains of equal molecular weight linked by disulfide bridges. Gel filtration of the purified antigen in the presence of Emulphogene suggested that it may be either a monomeric or a dimeric protein. However, in the presence of Triton X-100 a monomeric structure was implied. N-Glycanase digestion indicated that the protein is probably not glycosylated. The purified antigen was characterized as an integral membrane protein by hydrophobic chromatography and phase-separation experiments with Triton X-114. The antigen, or at least the antibody binding region of the molecule, is very susceptible to protease attack, as judged by protease digestion experiments on brain membranes. By using very low concentrations of papain combined with short incubation times, the antigen was converted to a 16.3-kDa membrane-associated polypeptide as assessed by immunoblotting. This polypeptide contained the BM88 binding epitope. Soluble BM88 immunoreactive polypeptides were not obtained. Bacillus cereus phospholipase C was also unable to solubilize the antigen from the membrane. Our results suggest that the molecule, possessing at least one small extramembranous domain, is attached to the membrane via a polypeptide chain.
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PMID:Purification and characterization of neuron-specific surface antigen defined by monoclonal antibody BM88. 170 20

A phosphatidylinositol-specific phospholipase C (PI-PLC) has been isolated from bovine brain (purification factor of 5.6 x 10(4)). By sodium dodecyl sulfate-polyacrylamide gel electrophoresis, it had a Mr of 57,000. Neither amino nor neutral sugars were detected in the purified enzyme. The pH optimum was 7.0-7.5, and the activity decreased only slightly at pH 8.0. When phosphatidylinositol was used as a substrate, the optimum Ca2+ requirement was 4 mM, and Km was 260 microM. When phosphatidylinositol 4,5-bisphosphate was used, the optimum Ca2+ requirement was 10(-7) M, and the Km was reduced to 90 microM. Lipid specificity studies showed that equal amounts of inositol phosphate and diacylglycerol were released from phosphatidylinositol but 4 times as much inositol 1,4,5-trisphosphate was released from phosphatidylinositol 4,5-bisphosphate. Other lipids, phosphatidylcholine, phosphatidylethanolamine, and sphingomyelin, were not substrates. Failure to detect phosphatidic acid confirmed the absence of a phospholipase D activity in the purified enzyme. Myelin basic protein (MBP) stimulated the PI-PLC activity between 2- and 3-fold. Histone had a small effect only, whereas bovine serum albumin and cytochrome C had no effect. Phosphorylation of MBP reduced the stimulatory effect. Protein-protein interactions between MBP and PI-PLC have been demonstrated both immunologically and by sucrose density gradients. A stoichiometry of 1:1 has been suggested by the latter method. A number of peptides have been prepared by chemical, enzymatic, and synthetic methods. Peptides containing the MBP sequences consisting of residues 24-33 and 114-122 stimulated the PI-PLC but were less effective than the intact protein.
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PMID:A 57-kDa phosphatidylinositol-specific phospholipase C from bovine brain. 170 49

K1 is a monoclonal antibody that reacts with a cell surface antigen (CAK1) found in human mesothelia and nonmucinous ovarian tumors. In this article, the characteristics of the CAK1 antigen have been examined in detail. Using immunofluorescence microscopy, we have found that the CAK1 signal is removed from the cell surface by treatment with proteases or by phosphatidylinositol-phospholipase C, but not by neuraminidase and beta-galactosidase. The phosphatidylinositol-phospholipase C-released material was found to contain the CAK1 antigen which was detected by a competition radioimmunoassay. The phosphatidylinositol-phospholipase C-released CAK1 antigen was examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting and found to be approximately 40 kDa protein. The CAK1-K1 antibody complex remains on the cell surface and is poorly internalized, as shown by an acid wash immunofluorescence internalization assay. An immunotoxin composed of K1 and Lys-PE40, a mutant form of Pseudomonas exotoxin lacking the cell binding domain, was not cytotoxic, supporting the conclusion that the CAK1-K1 antibody complex is not internalized. However, an immunotoxin composed of K1 and native Pseudomonas exotoxin was selectively cytotoxic to cells expressing the CAK1 antigen. This cytotoxicity is due to the fact that domain I of Pseudomonas exotoxin promotes internalization of antigens which are not internalized or bound to antibody alone. Our results suggest that CAK1 is a polypeptide that is expressed on mesothelial cells and many ovarian cancers, and that K1 may be useful as a targeting agent for the immunotherapy of human ovarian cancer.
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PMID:Characterization of the antigen (CAK1) recognized by monoclonal antibody K1 present on ovarian cancers and normal mesothelium. 172 78

The cellular receptor for human urokinase-type plasminogen activator (u-PAR) is shown by several independent criteria to be a true member of a family of integral membrane proteins, anchored to the plasma membrane exclusively by a COOH-terminal glycosyl-phosphatidylinositol moiety. 1) Amino acid analysis of u-PAR after micropurification by affinity chromatography and N-[2-hydroxy-1,1-bis(hydroxymethyl)-ethyl]glycine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed the presence of 2-3 mol of ethanolamine/mol protein. 2) Membrane-bound u-PAR is efficiently released from the surface of human U937 cells by trace amounts of purified bacterial phosphatidylinositol-specific phospholipase C. This soluble form of u-PAR retains the binding specificity toward both u-PA and its amino-terminal fragment holding the receptor-binding domain. 3) Treatment of purified u-PAR with phosphatidylinositol-specific phospholipase C or mild alkali completely alters the hydrophobic properties of the receptor as judged by temperature-induced detergent-phase separation and charge-shift electrophoresis. 4) Biosynthetic labeling of u-PAR was obtained with [3H]ethanolamine and myo-[3H]inositol. 5) Finally, comparison of amino acid compositions derived from cDNA sequence and amino acid analysis shows that a polypeptide of medium hydrophobicity is excised from the COOH terminus of the nascent u-PAR. A similar proteolytic processing has been reported for other proteins that are linked to the plasma membrane by a glycosyl-phosphatidylinositol membrane anchor.
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PMID:Cellular receptor for urokinase plasminogen activator. Carboxyl-terminal processing and membrane anchoring by glycosyl-phosphatidylinositol. 184 68

Phospholipase C was purified from human melanoma grown as solid tumors in nude mice. The specific activity of the pure enzyme was approx. 100 mumol/min per mg; its apparent molecular mass was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis to be 150 kDa. The enzyme required calcium for activity and was activated by deoxycholate in the presence of the substrate phosphatidylinositol. The melanoma phospholipase C has a distinctly different substrate preference than those identified from normal tissues; it prefers phosphatidylinositol to phosphatidylinositol bisphosphate. The tumor enzyme was approx. 4-5-fold more active using phosphatidylinositol than phosphatidylinositol bisphosphate as the substrate.
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PMID:Phospholipase C from human melanoma: purification and characterization of a phosphatidylinositol-selective enzyme. 184 29

Incubation of isolated cardiac myocytes from rat hearts with heparin or phosphatidylinositol-specific phospholipase C (PLC) resulted in the release of lipoprotein lipase (LPL) into the medium. The release of LPL by the combination of heparin and PLC was not additive, and preincubation of cardiac myocytes with heparin eliminated the release of LPL in a subsequent incubation with PLC. This evidence suggests that LPL may be bound ionically to heparan sulfate proteoglycans that are covalently linked to the cell surface of cardiac myocytes by a phosphatidylinositol-glycan membrane anchor; a second pool of LPL may also be bound to proteoglycans attached directly to the myocardial cell surface. The induction of diabetes by the administration of streptozotocin (100 mg/kg for 3-4 days) to rats resulted in a decrease in the initial cellular activity of LPL and a marked reduction in the heparin-induced secretion of LPL into the medium of cardiac myocytes. The intravenous administration of insulin (5 U for 1 h) in diabetic rats reversed the effects of diabetes on cellular and heparin-releasable LPL activities. Diabetes also reduced the PLC-induced release of LPL. The reduction in the release of LPL from diabetic cardiac myocytes could result in a decrease in functional LPL activity at the capillary endothelium of whole hearts.
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PMID:Diabetes reduces heparin- and phospholipase C-releasable lipoprotein lipase from cardiomyocytes. 184 7

Heterotrimeric GTP-binding proteins from bovine brain were resolved by fast protein liquid chromatography chromatography using Mono Q columns. Two distinct forms of the protein Go were identified. Both forms had stochiometric amounts of alpha- and beta gamma-subunits. The a-subunits of both forms were recognized by an alpha o-specific antiserum, but not by any of the alpha i-specific antisera. The two forms showed distinct migration patterns on 9% sodium dodecyl sulfate-polyacrylamide gels containing 4-8 M urea gradients. Neither form comigrated with the recombinant alpha o1. Both the recombinant alpha o1 and the most abundant form of Go were recognized by an antiserum, H-660, against a peptide encoding amino acids 3-17 of alpha i2. H-660 has been shown previously to recognize alpha o and alpha i (Mumby, S. M., Pang, I. K., Gilman, A. G., and Sternweis, P. C. (1988) J. Biol. Chem. 263, 2020-2026). This more abundant form is called Go A most likely corresponds to the cloned alpha o1. The less abundant form, Go B, was not recognized by H-660. However, both forms of bovine brain Go were recognized by GC/2, an antiserum against the N-terminal region of alpha o1. Hence alpha oA and alpha oB may be different in their N terminus regions. Neither form of bovine brain Go was recognized by an antisera made to a peptide encoding the unique regions of the cloned alpha o2 from HIT cells (Hsu W. H., Rudolph, U., Sanford, J., Bertrand, P., Olate, J., Nelson, C., Moss, L.E., Boyd, A. E., III, Codina, J., and Birnbaumer, L. (1990) J. Biol. Chem. 265, 11220-11226). Go A and Go B have similar guanine nucleotide binding and release properties. Both release GDP within 1 min in the absence of added Mg2+. Both bind guanosine (GTP gamma S) rapidly as well. However Go A binds GTP gamma S about 2.5-fold faster than Go B, in the absence of added Mg2+ ion. Both forms of Go as well as the recombinant alpha o (alpha o1) can increase muscarinic stimulation of inositol trisphosphate-mediated Cl- current in Xenopus oocytes. These data indicate that we have identified two structurally distinct forms of Go that have different guanine nucleotide binding properties and are capable of functioning in the receptor-regulated phospholipase C pathway in Xenopus oocytes.
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PMID:Two forms of the bovine brain Go that stimulate the inositol trisphosphate-mediated Cl- currents in Xenopus oocytes. Distinct guanine nucleotide binding properties. 185 56

The insoluble residue from Tetrahymena mimbres cells that had been preincubated in vivo for 2 h with [3H]myristic acid and then exhaustively delipidated with organic solvents retained radioactivity, principally in material which migrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent molecular mass of 10-14 kDa. This material was extractable from the delipidated cell residue with organic solvents known to solubilize phosphatidylinositol glycans (PI glycans). The same material could also be labeled with [3H]inositol, [14C]glucosamine, and [3H] ethanolamine. When the delipidated residue of cells labeled for 2 h with [3H]myristate was treated with phosphatidylinositol-specific phospholipase C or nitrous acid, much of the associated radioactivity was released. A similar release was obtained using the putative PI glycan fraction extracted from the cell residue. After further purification by thin layer chromatography, this latter material was hydrolyzed with HCl and shown to contain fatty acids, alkylglyceryl ethers, phosphate, inositol, glucosamine, mannose, and ethanolamine. The findings indicate that T. mimbres contains PI glycans resembling in structure those recently characterized in trypanosomes and mammalian cells. As the time of incubation with the radiotracers enumerated above was increased to 6-24 h, increasing amounts of radioactivity appeared in the 22-27-kDa region of sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels. This higher molecular weight material is shown in the companion paper (Pak, Y., Ryals, P.E., and Thompson, G.A., Jr. (1991) J. Biol. Chem. 266, 15054-15059) to be released by in vivo phosphatidylinositol-specific phospholipase C treatment. Thus T. mimbres contains a pool of free PI glycans and at least one phosphatidylinositol-anchored protein.
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PMID:Phosphatidylinositol-linked glycans and phosphatidylinositol-anchored proteins of Tetrahymena mimbres. 186 41


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