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)

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

Lymphocytes bearing the T-lymphocyte differentiation antigen RT6 play an important immunoregulatory role in the development of autoimmune diabetes in BB rats. Immunofluorescence studies suggest that diabetes-prone (DP)- but not diabetes-resistant (DR)-BB rat lymphocytes fail to express RT6 antigen during ontogeny. Two alloantigenic forms of the molecule exist, i.e., RT6.1 and RT6.2; both are linked to cell membranes by a phosphatidylinositol (PI) linkage. In these studies, PI-phospholipase C (PLC) treatment of lymphocytes from BB and normal rats followed by immunoabsorption and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of released proteins with anti-RT6 allotype-specific monoclonal antibodies was performed. RT6.1 in several nondiabetic rat strains was found to consist of a family of nonglycosylated and variably glycosylated molecules: an N-Glycanase-resistant 24,000- to 26,000-Mr peptide and four N-Glycanase-sensitive peptides of 29,000, 31,000, 33,000, and 34,000 Mr. In contrast, RT6.2 was found to be a 24,000- to 26,000-Mr nonglycosylated polypeptide. The electrophoretic pattern of RT6.1 was observed to be the same when the antigen was extracted from W3/25+ (CD4+) versus W3/25- T lymphocytes or from resting versus mitogen-activated cells. A pattern of bands characteristic of the RT6.1 antigen found in normal rat strains was detected after PLC treatment or detergent solubilization of lymphocytes obtained from DR rats. In contrast, no evidence of either RT6 species was found after PLC or detergent treatment of comparable numbers of T lymphocytes from DP-BB rats. Interestingly, T lymphocytes from Wistar-Furth (RT6.2+) x DP (RT6-) F1 crosses were observed to coexpress both RT6.2 and RT6.1 molecules, with the electrophoretic pattern of RT6.1 being similar to that obtained in DR and other rat strains. This study provides biochemical evidence that DP rats may have an intact RT6a structural gene.
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PMID:Biochemical studies of RT6 alloantigens in BB/Wor and normal rats. Evidence for intact unexpressed RT6a structural gene in diabetes-prone BB rats. 221 79

The pellet recovered after centrifugation (5000 X g) of human corneal endothelial homogenates was used as the source of membranes in these studies. A 66-kilodalton (kD) protein was identified as the most abundant protein in the particulate pellet by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The de novo synthesis of the 66-kD protein by endothelial cells was observed during culturing of human corneas in the presence of 35S-methionine. The 66-kD protein was found to be a plasma membrane protein based on several of its properties, ie, its solubility in CHCl3:CH3OH, its labeling as surface glycoprotein, and during exposure to a photoaffinity hydrophobic probe: 1-azido-4-125I-iodobenzene. Furthermore this protein could be released from the particulate pellet after treatment with phosphatidylinositol-specific phospholipase C, suggesting its anchorage via a phosphatidylinositol glycan linkage in the plasma membrane. Such anchorage of this protein was further confirmed by its labeling during culture of corneas in the presence of 3H-myoinositol. The glycoprotein nature of the 66-kD protein was evident from its labeling during surface glycoprotein labeling of endothelial cells, staining with periodic acid-Schiff stain, and binding to peanut agglutinin (PNA), and lotus agglutinin (LTA) on SDS-acrylamide gels. The 66-kD protein of endothelial particulate pellets recovered from corneas of donors of different ages showed an age-related increase in binding to PNA and LTA. This suggested an increased glycosylation of the 66-kD protein with aging. A polyclonal anti-66-kD protein antibody was used as a probe to determine the presence of this protein in the rabbit and bovine corneal endothelia by the Western-blot analysis. The 66-kD protein was detected in both rabbit and bovine endothelia, but an additional immunoreactive species of 17 kD was also observed which may be a processed product of the 66-kD protein.
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PMID:Characterization of a 66-kilodalton surface glycoprotein of the human corneal endothelium. 221 Sep 94

The terminal differentiation of Schwann cells is dependent on contact with basement membrane. The present study was undertaken to investigate the role of cell surface heparan sulfate proteoglycans (HSPGs) in mediating Schwann cell responses to extracellular matrix contact. Phosphatidylinositol-specific phospholipase C-releasable cell surface HSPGs purified from cultures of neonatal rat Schwann cells were subjected to affinity chromatography on immobilized laminin and fibronectin. Binding of the HSPG to both affinity matrices was observed. The strength of the association, however, was sensitive to the ionic strength of the buffer. In 0.1 M Tris-HCl, HSPG binding was essentially irreversible whereas in physiological ionic strength buffer (e.g. 0.142 M NaCl, 10 mM Tris), weaker binding was detected as a delay in elution of the HSPG from the affinity columns. Further studies of HSPG-laminin binding suggested that the binding was mediated by the glycosaminoglycan chains of the proteoglycans. Results of equilibrium gel filtration chromatography provided additional evidence for a reversible association of the HSPG and laminin with a Kd of approximately 1 x 10(-6) M. When Schwann cells were plated on plastic dishes coated with laminin, the cells attached and extended long slender processes. Inclusion of heparin, but not chondroitin sulfate, in the assay medium resulted in partial inhibition of process extension, but at concentrations of heparin which were higher than that needed to disrupt laminin-HSPG association in vitro. Addition of anti-integrin receptor antibodies resulted in more extensive inhibition of laminin-dependent process extension. Anti-integrin antibodies plus heparin essentially totally inhibited laminin-dependent process extension. These results demonstrate that cell surface HSPGs are capable of reversible association with extracellular matrix molecules and suggest that HSPG-laminin interactions play a role in laminin-dependent Schwann cell spreading.
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PMID:Association of cell surface heparan sulfate proteoglycans of Schwann cells with extracellular matrix proteins. 224 8

Rat liver 5'-nucleotidase was purified from a crude microsomal fraction, and its molecular mass was estimated to be 73 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified protein was subjected to cleavage with CNBr or lysyl endopeptidase, and the resulting 21 peptides as well as the NH2 terminus of the native protein were sequenced by Edman degradation. For further information on the molecular structure, we constructed a lambda gt11 liver cDNA library and isolated two cDNA clones for 5'-nucleotidase, lambda cNTP6 and lambda cNT34. The 3.2-kilobase cDNA insert of lambda cNTP6 contains an open reading frame that encodes a 576-residue polypeptide with a calculated size of 63,965 Da, which is in reasonable agreement with that of 5'-nucleotidase (62 kDa) immunoprecipitated from cell-free translation products. The NH2-terminal 28 residues comprise a signal peptide, which is followed by the NH2-terminal sequence of the purified protein. The predicted structure contains all the other peptide sequences determined by Edman degradation. Five potential N-linked glycosylation sites are found in the molecule, accounting for the difference in mass between the precursor and mature forms. Another characteristic feature is that the primary structure contains a highly hydrophobic amino acid sequence at the COOH terminus, a possible signal for the post-translational modification by glycophospholipid. In fact, labeling experiments of rat hepatocytes demonstrated that 3H-labeled compounds such as ethanolamine, myo-inositol, and palmitic acid, components of the glycolipid anchor, were incorporated into 5'-nucleotidase. Phosphatidylinositol-specific phospholipase C released 5'-nucleotidase from the cell surface, and the released protein no longer contained the radioactivity of [3H]palmitic acid incorporated.
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PMID:Primary structure of rat liver 5'-nucleotidase deduced from the cDNA. Presence of the COOH-terminal hydrophobic domain for possible post-translational modification by glycophospholipid. 229 43

alpha-Thrombin and phorbol 12,13-dibutyrate stimulated the mono(ADP-ribosyl)ation of a 42-kDa cytosolic protein of human platelets. This effect was mediated by protein kinase C activation and was inhibited by protein kinase C inhibitor staurosporine. It also was prevented by prostacyclin, which is known to inhibit the phospholipase C-induced formation of 1,2-diacylglycerol, which is one of the endogenous activators of protein kinase C. On sodium dodecyl sulfate/polyacrylamide gel electrophoresis, the 42-kDa protein that is ADP-ribosylated by alpha-thrombin was clearly distinct from the alpha subunits of membrane-bound inhibitory and stimulatory guanine nucleotide-binding regulatory proteins, respectively Gi alpha and Gs alpha; the 47-kDa protein that is phophorylated by protein kinase C in platelets; and the 39-kDa protein that has been shown to be endogenously ADP-ribosylated by agents that release nitric oxide. This information shows that agonist-induced activation of protein kinase leads to the ADP-ribosylation of a specific protein. This covalent modification might have a functional role in platelet activation.
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PMID:Agonist-induced ADP-ribosylation of a cytosolic protein in human platelets. 233 84

Evidence presented demonstrates a covalent attachment of a phospholipid to bovine myelin basic protein. Partial characterization of the phospholipid moiety was performed on myelin basic protein obtained from 32P-phosphorylated whole myelin that was first delipidated by two ether/ethanol (3:2 v/v) extractions, ether extraction, and acetone extraction and then purified by preparative sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The myelin basic protein was precipitated with aqueous acetone and treated with proteases. Treatment with carboxypeptidase Y or trypsin for several hours released a lipophilic fragment, which was purified by reverse-phase high-performance liquid chromatography to yield two "lipopeptides". Such lipopeptides were obtained from both the major and minor myelin basic proteins of rat and bovine brain. Treatment with either mild base or phospholipase C removes the lipophilic character of the peptide fragment. The lipophilic fragment is a substrate for phospholipase D, but it does not comigrate on thin-layer chromatography with any 32P-labeled lipid obtained from myelin incubated with [gamma-32P]ATP. Polyphosphoinositides were shown to be released by mild acid treatment of myelin basic protein that had been extracted with organic solvent and then purified by SDS-polyacrylamide gel electrophoresis. Along with the fact that inositol monophosphate was identified in the partial acid hydrolysate of the lipopeptide, we have concluded that polyphosphoinositide (phosphatidylinositol 4-phosphate and/or phosphatidylinositol 4,5-bisphosphate) was the original phospholipid portion of the lipopeptide.
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PMID:Colvalent linkage of phospholipid to myelin basic protein: identification of phosphatidylinositol bisphosphate as the attached phospholipid. 242 99

Intact bloodstream forms of Trypanosoma brucei brucei, T.b. gambiense, and T.b. rhodesiense and procyclic forms of T.b. brucei and T.b. gambiense were incubated in trypsin, solubilized for gel electrophoresis, and analyzed for removal of surface molecules. Silver-stained gels and transfer blots probed with horseradish peroxidase-conjugated or radiolabeled lectins revealed that only three glycoproteins, Gp120p, Gp91p, and Gp23p, were removed from the surface of procyclic forms by trypsin. The variant specific glycoproteins, Gp23b, Gp120b, and in some clones Gp91b were surface molecules cleaved from bloodstream forms. Greater than 90% of the variant specific glycoprotein (VSG) was removed from the surface of all clones studied within 1 hr following the addition of trypsin. The removal of VSG was coincident with appearance of 37 to 50 kDa glycopeptide fragments of VSG with different clones yielding different sized fragments. Detailed kinetic analysis of proteins from whole cell extracts and supernatants of the DuTat 1.1 clone of T.b. rhodesiense using concanavalin A (Con A) and polyclonal antibodies revealed that three major VSG fragments were released during trypsinization. The electrophoretic mobility of the three VSG fragments of DuTat 1.1 was not altered when samples were boiled in sodium dodecyl sulfate to inhibit the endogenous phospholipase C. Antiserum to the cross-reactive determinant bound to intact VSG, but did not bind VSG fragments. Thus, the major Con A binding fragments of DuTat 1.1 VSG and perhaps those of the other clones we studied were probably derived from the N-terminal domain of the molecule. The data suggest that VSG is cleaved by trypsin in situ at the hinge region, but remains attached to the cell surface via weak interaction with neighboring molecules.
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PMID:Trypanosoma brucei sspp.: cleavage of variant specific and common glycoproteins during exposure of live cells to trypsin. 245 21

The mouse lymphocyte surface alloantigen, Ly-31, defined by monoclonal antibody N1.10 (IgG2b,k) and controlled by a gene locus closely linked to the Akp-2 locus on chromosome 4, was biochemically investigated. By employing a quantitative immunoassay system, it was found that the Ly-31.1-specific antibody detected an allotypic determinant of mouse alkaline phosphatase. Ly-31.1, i.e., mouse alkaline phosphatase, was expressed predominantly in kidney and bone and was also detected in placenta, lung, and testis. Concerning tumor cell lines, they varied in the amount of antigen present, with both T and B lymphoid lineages selectively possessing the antigen. In normal lymphoid tissues, lesser amounts of antigen were detected. The binding of mouse alkaline phosphatase to Ly-31.1-specific monoclonal antibodies was specific in nature. The Ly-31.1 antigen was immunoprecipitated from the lysates of surface-radiolabeled YAC-1 moloney leukemia cells, and appeared as a single band of about 78,000 under both reduced and nonreduced conditions on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Furthermore, treatment of tumor cell lines with phosphatidylinositol-specific-phospholipase C resulted in the removal of Ly-31 antigen from the cell surface. These results suggest that a gene cluster containing the Ly-31 and Akp-2 loci which control the alkaline phosphatase is formed on mouse chromosome 4. The Ly-31 antigen is the first enzyme demonstrated to be a lymphocyte surface alloantigen.
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PMID:Mouse Ly-31.1 is an alloantigenic determinant of alkaline phosphatase predominantly expressed in the kidney and bone. 246 81

Phosphatidylinositol-specific phospholipase C was purified in a 27% yield from the culture medium of Bacillus cereus by a combination of ammonium sulfate precipitation and ion-exchange and hydrophobic interaction chromatography. The purified enzyme was free of other phospholipase C-type activities and exhibited a high specific activity of approximately 1,300 units/mg. Amino acid composition analysis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated a molecular weight of about 35 kDa. The sequence of the first 29 N-terminal amino acids was also determined.
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PMID:Phosphatidylinositol-specific phospholipase C from Bacillus cereus: improved purification, amino acid composition, and amino-terminal sequence. 249 36


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