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

A genomic clone was isolated which codes for the fructose bisphosphate aldolase of Plasmodium falciparum. The aldolase gene is interrupted by one intron which divides the coding region into two exons. The first one codes for one amino acid only, the initiation methionine, while the second one encodes the residual 368 amino acids of the protein. The gene, which is represented only once in the genome, is transcribed at high rates as a 2.4-kb mRNA in the P. falciparum blood stage. The aldolase gene encodes a protein of 40,105 Da, which is 61-68% homologous to known eukaryotic aldolases. The protein was expressed in Escherichia coli cells in an unfused and enzymatically active form. Antisera raised against amino acids 9-96 recognize a 41-kDa protein band previously shown to protect monkeys against a P. falciparum infection. These antisera cross-react with aldolases of different species, which confirms the strong conservation of this enzyme during evolution. The aldolase could be localized in the cytoplasm of the parasite as an active and soluble form. An inactive form was found to be associated with the membrane fraction. Digestion data with phospholipase C suggest a membrane association of this polypeptide via a glycosylphosphatidylinositol anchor.
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PMID:Plasmodium falciparum aldolase: gene structure and localization. 219 85

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

Complementary DNA clones coding for both carcinoembryonic antigen (CEA), a well characterized colonic tumor marker, and nonspecific cross-reacting antigen (NCA), a related antigen, were expressed in Chinese hamster ovary (CHO) cells and L-cells (mouse fibroblasts). A genomic clone coding for CEA was also expressed in CHO cells. Positive clones were identified by fluorescence flow cytometry and enzyme-linked immunosorbent assay. Membrane location of the recombinant CEA and NCA was confirmed by indirect immunofluorescence labeling of the transfectants, followed by visualization under a fluorescence microscope. The apparent molecular weight of the expressed CEA and NCA were 180,000 and 96,000, respectively, for both cell lines, as determined by immunoblot analysis. The CEA and NCA expressed on CHO cells were sensitive to treatment with phosphatidylinositol-specific phospholipase C (PI-PLC), whereas the CEA and NCA proteins on L-cells were resistant to removal by PI-PLC. Unlike NCA, which contains three methionine residues, the only methionine in CEA is in the C-terminal hydrophobic domain. This domain in CEA was shown to be removed and replaced by a phosphatidylinositol glycan (PI-G) anchor (Hefta et al., Proc. Natl. Acad. Sci. USA, 85: 4648-4652, 1988). The recombinant CEA from both CHO cells and L-cells could be labeled with [3H]-ethanolamine (a component of the PI-G anchor) but not with [35S] methionine, whereas the recombinant NCA could be labeled with both [3H]ethanolamine and [35S]methionine. The labeling studies and PI-PLC treatment results are consistent with the CEA and NCA expressed on CHO cells possessing a PI-G anchor. The CEA expressed on the L-cell transfectants may contain a PI-G anchor which is resistant to cleavage by PI-PLC. In addition, the membrane-bound and secreted levels of CEA from the CHO and L-cell transfectants were determined.
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PMID:Expression of complementary DNA and genomic clones for carcinoembryonic antigen and nonspecific cross-reacting antigen in Chinese hamster ovary and mouse fibroblast cells and characterization of the membrane-expressed products. 231 24

In neutrophils and several other phagocytes, a pertussis and cholera toxin-sensitive guanine nucleotide-binding protein (G-protein) couples the receptors for formyl methionine-containing chemotactic peptides to stimulation of phospholipase C. We used membranes of myeloid-differentiated HL 60 cells to study the role of Na+ in regulating both the interaction of the formyl peptide receptor with the chemotactic agonist, N-formyl-methionyl-leucyl-phenylalanine (FMLP), and the receptor-mediated activation of the G-protein. Monovalent cations (Na+ greater than Li+ greater than K+ greater than choline+) markedly inhibited the binding of the radiolabeled oligopeptide [3H]FMLP by specifically reducing the number of receptors in the high-affinity state. Half-maximal and maximal inhibition of peptide binding were seen at cation concentrations of approximately 20 and 200 mM, respectively. Inhibition of peptide binding by Na+ was observed in the presence and absence of divalent cations and was strictly additive to inhibition by the poorly hydrolyzable GTP analogue, guanosine-5'-O-(3-thiotriphosphate), or to ADP ribosylation of G-proteins by pertussis toxin. The inhibitory effect of Na+ on peptide binding coincided with a marked reduction of the potency of FMLP to stimulate a high-affinity GTPase. In contrast, the degree of FMLP-stimulated GTPase activity was markedly enhanced in the presence of Na+. This was largely due to the fact that Na+ reduced the agonist-independent basal GTPase activity in the same way but less so than pertussis toxin treatment. The results show that monovalent cations, Na+ in particular, regulate the interaction of the formyl peptide receptor with both the chemotactic agonist and the G-protein by acting on a single site, possibly located on the receptor itself. The observation that basal GTPase activity is markedly reduced by both Na+ and pertussis toxin treatment also suggests (a) that G-proteins interact with and are activated by receptors even in the absence of agonists and (b) that Na+ uncouples unoccupied receptors from G-protein interaction and activation.
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PMID:Na+ regulation of formyl peptide receptor-mediated signal transduction in HL 60 cells. Evidence that the cation prevents activation of the G-protein by unoccupied receptors. 251 70

Both phosphatidylethanolamine(PE)-N-methylation and phosphatidyl-inositol bisphosphate (PI-bisphosphate) breakdown potentially modify the microdomains in the sarcolemmal lipid bilayer. In this study the possibility of a mutual interaction between the enzymes responsible for these phospholipid reactions is examined. In sarcolemma purified from rat heart, prior hydrolysis of PI lipids by exogenous specific phospholipase C inhibited (to 75, 59 and 78% of control for sites I, II and II, respectively) the PE-N-methyltransferase system. In cultured rat cardiomyocytes the addition of L-methionine, a precursor for the methyl donor S-adenosylmethionine, stimulated PE-N-methylation in a concentration (0.2-300 microM)-dependent manner. Methionine (50 microM) decreased the basal rate of PI-bisphosphate hydrolysis (to 72% of control), but had no effect on the phenylephrine-stimulated PI-bisphosphate hydrolysis. Maximal activation of the PI-bisphosphate breakdown by 30 microM phenylephrine did not affect the rate of PE-N-methylation in the presence of exogenous methionine (50 microM). These findings support the existence of interactions, although discrete, between the enzymes involved in the PE-N-methylation and PI turnover.
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PMID:Discrete interactions between phosphatidylethanolamine-N-methylation and phosphatidylinositolbisphosphate hydrolysis in rat myocardium. 257 24

Neutrophils pretreated with phorbol 12-myristate 13-acetate (1-10 nM) and stimulated with low concentrations of chemotactic agonists (1-10nM) exhibited a marked increase in respiratory burst activity that was characterized by regular oscillations. These were accompanied by parallel oscillations in turbidity having the same phase and period. Four different agonists, f-Met-Leu-Phe, complement fragment C5a, platelet-activating factor, and leukotriene B4, induced virtually identical oscillations, with mean periods of 7.9 +/- 0.6 s (respiratory burst) and 7.9 +/- 0.8 s (turbidity) at 37 degrees C. No burst oscillations were observed at high agonist concentrations (50-100 nM) unless the fungal metabolite 17-hydroxywortmannin was added prior to stimulation. In the absence of phorbol 12-myristate 13-acetate, the respiratory burst activity was inhibited by 17-hydroxywortmannin, the protein kinase C inhibitor staurosporine, and calcium depletion, while agonist-dependent turbidity changes including the oscillations were unaffected. Turbidity changes reflect corresponding changes in cell size and/or shape, suggesting that cyclic alterations in morphology such as lamellipod extension and retraction physically affect the catalytic efficiency of the membrane-bound burst enzyme NADPH-oxidase. The oscillations appear to be controlled via receptor-dependent activation mechanisms which do not involve PKC activation or the rise in internal calcium presumably derived from phospholipase C activation.
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PMID:Respiratory burst oscillations in human neutrophils and their correlation with fluctuations in apparent cell shape. 277 66

We have investigated the post-translational modification of carcinoembryonic antigen (CEA) for membrane-anchoring in QGP-1 cells derived from a human pancreatic carcinoma. Pulse-chase experiments with [3H]leucine demonstrated that CEA was initially synthesized as a precursor form with Mr 150,000 having N-linked high-mannose-type oligosaccharides, which was then converted to a mature form with Mr 200,000 containing the complex type sugar chains. The mature protein thus labeled was found to be released from the cell surface by treatment with phosphatidylinositol-specific phospholipase C, suggesting that CEA is a phosphatidylinositol-linked membrane protein. This was confirmed by metabolic incorporation into CEA of 3H-labeled compounds such as ethanolamine, myo-inositol, palmitic acid, and stearic acid. The 3H-labeled fatty acids incorporated were specifically removed from the protein by nitrous acid deamination as well as by phosphatidylinositol-specific phospholipase C treatment. Since the available cDNA sequence predicts that CEA contains a single methionine residue only in its carboxyl-terminal hydrophobic domain, processing of the carboxyl terminus was examined by pulse-chase experiments with [35S]methionine. It was found that CEA with Mr 150,000 was initially labeled with [35S]methionine but its radioactivity was immediately lost with chase. Taken together, these results suggest that CEA is anchored to the membrane by simultaneously occurring proteolysis of the carboxyl terminus and replacement by the glycophospholipid immediately after the synthesis.
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PMID:Evidence for carboxyl-terminal processing and glycolipid-anchoring of human carcinoembryonic antigen. 284 40

Epidermal growth factor (EGF) stimulated the rapid accumulation of inositol trisphosphate in WB cells, a continuous line of rat hepatic epithelial cells. Since we previously had shown that EGF stimulates EGF receptor synthesis in these cells, we tested whether hormones that stimulate PtdIns(4,5)P2 hydrolysis would increase EGF receptor protein synthesis and mRNA levels. Epinephrine, angiotensin II, and [Arg8]vasopressin activate phospholipase C in WB cells as evidenced by the accumulation of the inositol phosphates, inositol monophosphate, inositol bisphosphate, and inositol trisphosphate. A 3-4-h treatment with each hormone also increased the rate of EGF receptor protein synthesis by 3-6-fold as assessed by immunoprecipitation of EGF receptor from [35S]methionine-labeled cells. Northern blot analyses of WB cell EGF receptor mRNA levels revealed that agents linked to the phosphoinositide signaling system increased receptor mRNA content within 1-2 h. A maximal increase of 3-7-fold was observed after a 3-h exposure to EGF and hormones. The phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA), which activates protein kinase C also stimulated EGF receptor synthesis. Pretreatment of WB cells for 18 h with high concentrations of TPA "down-regulated" protein kinase C and blocked TPA-directed EGF receptor mRNA synthesis. In contrast, the effect of EGF on EGF receptor mRNA levels was not significantly decreased by TPA pretreatment. Epinephrine-induced increases in EGF receptor mRNA were reduced from 4- to 2-fold. Similarly, 18 h TPA pretreatment abolished the effect of TPA on EGF receptor protein synthesis but did not affect EGF-dependent EGF receptor protein synthesis. The 18-h TPA pretreatment diminished by 30-50% the induction of receptor protein synthesis by epinephrine or angiotensin II. We conclude that in WB cells EGF receptor synthesis can be regulated by EGF and other hormones that stimulate PtdIns(4,5)P2 hydrolysis. In these cells, EGF receptor synthesis appears to be regulated by several mechanism: one pathway is dependent upon EGF receptor activation and can operate independently of protein kinase C activation; another pathway is correlated with PtdIns(4,5)P2 hydrolysis and is dependent, at least in part, upon protein kinase C activation.
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PMID:Epidermal growth factor (EGF) and hormones stimulate phosphoinositide hydrolysis and increase EGF receptor protein synthesis and mRNA levels in rat liver epithelial cells. Evidence for protein kinase C-dependent and -independent pathways. 284 41

The effects of 17-hydroxywortmannin (HWT), a powerful inhibitor of the respiratory burst associated with phagocytosis (Baggiolini, M., Dewald, B., Schnyder, J., Ruch, W., Cooper, P. H., and Payne, T. G. (1987) Exp. Cell Res. 169, 408-418), were studied in human neutrophils stimulated with chemotactic agonists or phorbol myristate acetate. At nanomolar concentrations HWT inhibited superoxide production and the release of granule contents induced by N-formyl-Met-Leu-Phe, C5a, platelet-activating factor, and leukotriene B4, but not by phorbol myristate acetate, indicating that it interferes with receptor-mediated activation of the neutrophils, without directly affecting protein kinase C (Ca2+/phospholipid-dependent enzyme), the NADPH-oxidase, or the process of granule exocytosis. Moreover, HWT did not influence agonist-induced [Ca2+]i changes, indicating that it does not interfere with the function of agonist receptors, G-proteins or the phosphatidylinositol-specific phospholipase C. By studying the effect of HWT on the respiratory burst elicited in normal and Ca2+-depleted cells by combined stimulation with N-formyl-Met-Leu-Phe and phorbol myristate acetate, evidence was obtained that two transduction sequences, both of which are G-protein-dependent, are necessary for the induction of the response by receptor agonists. One sequence is Ca2+-dependent, HWT-insensitive, and leads to activation of protein kinase C, the other is Ca2+-independent and HWT-sensitive. Ca2+ depletion, which blocks the first, and HWT, which blocks the second, can be used to show that both processes must be functional for the transduction of agonist signals into a respiratory burst response.
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PMID:Two transduction sequences are necessary for neutrophil activation by receptor agonists. 284 36


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