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 characteristics of the specific binding of labelled insulin to turtle thyroid microsomes were investigated. Binding experiments were performed in Krebs-Ringer bicarbonate buffer (pH 7.4) at 25 or 4 degrees C for different periods of time. Dissociation of the labelled insulin from the binding sites was also evaluated. It was found that the binding is dependent on time, temperature and microsomal protein concentration, with an optimum pH of 8.0. Unlabelled insulin and pro-insulin competed with the labelled insulin, binding in direct proportion to their biological activities, while glucagon and growth hormones did not compete for the binding sites. Scatchard plot analysis established the presence of binding sites of high and low affinities, and the rate of dissociation of bound insulin was considerably increased by the addition of unlabelled insulin. Both results are compatible with a negative co-operativity site-site interaction model. Trypsin abolished the insulin binding. These findings indicate that the microsomes from the turtle thyroid gland contain specific binding sites for insulin. However, pre-incubation of microsomes with phospholipase C or S-adenosyl-L-methionine (SAM), or incubation in the presence of 2 mol NaCl/l did not increase the specific insulin binding. Therefore, the binding properties are similar to those observed in mammalian insulin-responsive tissues except for the absence of the effects of 2 mol NaCl/l, phospholipase C or SAM, which suggests the absence of masked insulin-binding sites.
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PMID:Characterization of the insulin-binding sites in turtle thyroid microsomes. 351 Nov 68

The neural cell adhesion molecule (N-CAM) of rodents comprises three distinct proteins of Mr 180,000, 140,000, and 120,000 (designated N-CAM-180, -140, and -120). They are expressed in different proportions by different tissues and cell types. but the individual contribution of each form to cell adhesion is presently unknown. Previous studies have shown that the two N-CAM species of higher relative molecular mass span the membrane whereas N-CAM-120 lacks a transmembrane domain and can be released from the cell surface by phosphatidylinositol-specific phospholipase C. In this report, we provided evidence that N-CAM-120 contained covalently bound phosphatidylinositol and studied N-CAM-120 from its biosynthesis to its membrane insertion and finally to its release from the cell surface. Evidence was presented showing that the lipid tail of N-CAM-120 contained ethanolamine as is the case for other lipid-linked molecules. The phospholipid anchor was attached to the protein during the first minutes after completion of the polypeptide chain. This process took place in the endoplasmic reticulum as judged from endoglycosidase H digestion experiments. Immediately after a 2-min pulse with [35S]methionine, we detected also a short-lived precursor that had not yet acquired the lipid tail. Pulse-chase studies established that N-CAM-120 was transported to the cell surface from which it was slowly released into the extracellular milieu. The molecules recovered in the incubation medium appeared to have lost all of their bound fatty acid but only around half of the ethanolamine. Upon fractionation of brain tissue, approximately 75% of N-CAM-120 was recovered with a membrane fraction and approximately 25% in a membrane-free supernatant. A small proportion (approximately 6%) was found to be resistant to extraction by non-ionic detergent. A major posttranslational modification of N-CAM is polysialylation. Our results showed that also N-CAM-120 was polysialylated in the young postnatal brain and released in this form from cultured cerebellar cells. The presence of N-CAM in a form that can be released from the cell surface and accumulates in the extracellular fluid suggests a novel mechanism by which N-CAM-mediated adhesion may be modulated.
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PMID:Biosynthesis, membrane association, and release of N-CAM-120, a phosphatidylinositol-linked form of the neural cell adhesion molecule. 369 91

The specific binding of [125I]insulin to submaxillary gland microsomes was significantly enhanced by increasing the ionic strength of the incubation medium. This effect was neither related to changes in receptor or hormone degradation nor in the polymerization of the tracer. When equilibrium binding data from competition curves of unlabelled insulin versus [125I]insulin were analyzed, a marked increase in total binding capacity in high ionic strength was observed (from 890 to 2440 fmol/mg protein), with no change in binding affinity. Phospholipase C digestion was also able to increase specific [125I]insulin binding to microsomes. These results suggest the presence of masked receptors in submaxillary gland microsomes. Methylation of rat submaxillary gland microsomes by using S-adenosyl-L-methionine as the methyl donor significantly increased [125I]insulin binding. Scatchard analysis of the equilibrium binding data showed that addition of S-adenosyl-L-methionine (0.46 mM) to microsomes resulted in an enhancement of the total binding capacity (from 990 to 1520 fmol/mg protein) with no change in the affinity constants, which suggests the exposure of masked insulin receptors under such conditions. Both the methyl group incorporation into membrane phospholipids and the effect on insulin binding were dependent on the S-adenosyl-L-methionine concentration used and were partially suppressed in the presence of S-adenosyl-L-homocysteine, a specific competitive inhibitor of the methyltransferases activity. When microsomes were treated with S-adenosyl-L-[methyl-3H]methionine, the 3H-labelled methyl groups incorporated were found mainly in the lipid fraction associated to phosphatidylcholine, suggesting in this case that the unmasking of insulin receptors could be a consequence of alterations produced in membrane composition. The effects of phospholipase C, S-adenosyl-L-methionine and high ionic strength on insulin binding were not additive, suggesting that these procedures unmask receptors from the same pool.
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PMID:Unmasking of insulin receptors in rat submaxillary gland microsomes: effect of high ionic strength, phospholipase C and S-adenosyl-L-methionine. 389 Sep 58

Incubation of human neutrophils with phospholipase C from Clostridium perfringens caused an increase in the ability of the treated cells to bind the chemotactic peptide, F-Met-Leu-Phe. The increase in binding was related to an increase in specific binding of the ligand. The increase in specific binding was, in turn, related to an increased number of peptide receptors. The dissociation constant (KD) for the tripeptide was not altered, on the average, by enzyme treatment. The increase in peptide receptor number was related temporally, and possibly mechanistically, to enzyme-stimulated secretory function involving the secondary granules. Phospholipase C treatment did not similarly augment binding of the complement-derived attractant, C5a. Receptor numbers for different chemotactic ligands may therefore be controlled by different mechanisms. Supplementary experiments provided evidence that this phenomenon was attributable to phospholipase C activity and not to contaminating protease(s).
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PMID:Human neutrophil peptide receptors: mobilization mediated by phospholipase C. 628 27

Recently we described a saturable, high-affinity binding site for vesicular stomatitis virus (VSV) on the surface of Vero cells that appears to mediate viral infectivity. To isolate this binding site, we have extracted Vero cells with the detergent, octyl-beta-D-glucopyranoside. The dialyzed detergent extract specifically inhibits the saturable, high-affinity binding of 35S-methionine-labeled VSV to Vero cells. The inhibitory activity is resistant to protease, neuraminidase and heating to 100 degrees C. It is soluble in chloroform-methanol and inactivated by phospholipase C, suggesting that it is a phospholipid. Of various purified lipids tested, only phosphatidylserine was capable of totally inhibiting the high-affinity binding of VSV. The half-maximal inhibitory concentration for phosphatidylserine was 1 microM. Phosphatidylserine also inhibited VSV plaque formation by 80%-90%; Herpes simplex virus plaque formation was unaffected. Centrifugation and electron microscopy studies have shown that phosphatidylserine-containing liposomes bind to VSV. The finding that phosphatidylserine directly binds to VSV and inhibits VSV attachment and infectivity suggests that plasma membrane phosphatidylserine could function as a binding site or portion of a binding site for VSV.
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PMID:Inhibition of VSV binding and infectivity by phosphatidylserine: is phosphatidylserine a VSV-binding site? 629 4

Phallolysin, a mixture of two to three cytolytic proteins (all of Mr 34 000), has been isolated from Amanita phalloides mushrooms and purified to homogeneity (specific activity 24 000 hemolytic units/mg of protein). After separation by isoelectric focusing, the amino acid composition of two of these proteins has been determined. They are rich in water-soluble amino acids and contain one tryptophan residue each, but no cysteine or methionine. Mr was determined to be 34 000 in the native form as well as under denaturing conditions, indicating that the native proteins exist as monomers. Many of the physical properties of phallolysin are strikingly similar to those of staphylococcal alpha-toxin, e.g., molecular weight, existence of multiple forms, pI values, amino acid composition, and thermolability (60 degrees C). Pure phallolysin allowed us to prepare a radioactively labeled toxin. Labeling was achieved by reaction with formaldehyde, followed by reduction with sodium [3H]borohydride. With the labeled toxin (specific activity 7-14 Ci/mmol, ca. 60% biological activity), we investigated its binding to human A2 erythrocytes. We determined the number of receptors on these cells (2 X 10(4) per cell) as well as their affinity to the toxin (KD = 4 X 10(-9) M). In studies on the mechanism of cytolytic activity, we were able to distinguish at least three sequential events: binding of the toxin to human erythrocytes, K+ release, and membrane rupture (hemoglobulin release). These steps could be characterized by different kinetics as well as by different temperature dependencies. Again, the kinetic data for phallolysin are very closely related to those obtained for staphylococcal alpha-toxin.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Physical properties and function of phallolysin. 662 15

Phosphatidylethanolamine of rat liver microsomes is rapidly methylated by S-adenosyl[methyl-14C]methionine to produce phosphatidyl-N-monomethylethanolamine, phosphatidyl-N,N-dimethylethanolamine and phosphatidylcholine. Using phospholipase C as a probe, on both opened (0.4% taurocholate or French pressure cell treatment) and unopened microsomes, it is demonstrated that phosphatidylcholine is labelled in the inner leaflet of the bilayer and, to a greater extent, in the outer leaflet. Phosphatidyl-N,N-dimethylethanolamine is labelled in the outer leaflet and in a pool sequestered from phospholipase C in open and closed vesicles. Phosphatidyl-N-monomethylethanolamine is labelled in a similarly sequestered pool. When microsomes containing labelled phosphatidyl-N-monomethylethanolamine and phosphatidyl-N,N-dimethylethanolamine were incubated with unlabeled S-adenosylmethionine, these phospholipids were methylated to produce phosphatidylcholine in the outer leaflet. This metabolism was inhibited by S-adenosylhomocysteine. Trypsin treatment of unopened microsomes inhibited 95% of the incorporation of 14CH3 into the outer leaflet of the bilayer with no effect on incorporation into sequestered phosphatidyl-N-monomethylethanolamine, phosphatidyl-N,N-dimethylethanolamine and phosphatidylcholine. Therefore, sequestered phosphatidyl-N-monomethylethanolamine and phosphatidyl-N,N-dimethylethanolamine are apparently synthesized by enzymes located at the inner surface of the microsomal membranes. These observations suggest that initial methylation of phosphatidylethanolamine takes place at the inner surface of the microsomes and that phosphatidyl-N-monomethylethanolamine is transferred to the outer leaflet to produce phosphatidylcholine. However, phosphatidyl-N-monomethylethanolamine is also methylated at the inner leaflet to produce phosphatidylcholine which does not equilibrate with that of the outer leaflet. Phosphatidylcholine of both the inner and outer bilayer leaflets is uniformly labelled by injection of [14C]methionine, in vivo.
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PMID:Biogenesis of endoplasmic reticulum phosphatidylcholine. Translocation of intermediates across the membrane bilayer during methylation of phosphatidylethanolamine. 721 79

Hepatocyte growth factor/scatter factor (HGF/SF) is a multifunctional cytokine that induces mitogenesis, motility, invasion, and morphogenesis of several epithelial and endothelial cell lines in culture. The receptor for HGF/SF has been identified as the Met tyrosine kinase. To investigate the signaling pathways that are involved in these events, we have generated chimeric receptors containing the extracellular domain of the colony-stimulating factor-1 (CSF-1) receptor fused to the transmembrane and intracellular domains of the Met receptor (MET). Madin-Darby canine kidney (MDCK) epithelial cells expressing the CSF-MET chimera dissociate and scatter in response to CSF-1. However, cells expressing a mutant CSF-MET receptor containing a phenylalanine substitution for tyrosine 1356 were unable to scatter or form branching tubules following stimulation with CSF-1. Tyrosine 1356 is essential for the recruitment of multiple substrates including the p85 subunit of PI3-kinase, phospholipase C gamma, and Grb2. In this study, we have investigated the role of PI3-kinase and a downstream target of PI3-kinase, pp70S6K, in the induction of MDCK cell scatter in response to HGF/SF. Our results demonstrate that following stimulation with HGF/SF, activation of PI3-kinase but not pp70S6K is essential for MDCK cell scatter.
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PMID:Hepatocyte growth factor-induced scatter of Madin-Darby canine kidney cells requires phosphatidylinositol 3-kinase. 749 47

Chronic blockade of opioid receptors by naltrexone increases opioid peptides in the striatum, and up-regulates brain opioid receptors resulting in functional supersensitivity. Striatal SP content was increased 3.5-fold after 8 days of naltrexone treatment relative to control animals. The present study was undertaken to determine whether SP receptors in the striatum and SP receptor-coupled second messenger system are modulated by increased striatal SP content induced by chronic opioid receptor blockade. The binding affinity and capacity of SP receptors, determined using [125I]Bolton-Hunter SP ([125I]BHSP) labeled at Lys3, in striatal synaptosomal membranes were not significantly altered by chronic blockade of opioid receptors. Although the concentrations of [Sar9,Met (O2)11]SP, a NK-1 receptor-specific agonist, and SP(1-7), an aminoterminal major metabolite of SP, required to inhibit half of [125I]BHSP binding (IC50) in striatal synaptosomal membranes were significantly decreased, the IC50s for SP and an NK-2 receptor-specific agonist, [Nle10]NK A (4-10), remained unchanged by chronic naltrexone treatment. The data suggest that naltrexone which has no SP receptor antagonistic action, not only indirectly acts on SP-ergic neurons but also causes a change in the apparent affinity of NK-1 receptor (as reflected by changes in IC50 values) in the striatum. Cellular inositol-1,4,5-trisphosphate [Ins(1,4,5)P3], quantified by a highly sensitive and selective radioreceptor mass assay, was increased in the striatum by 28% relative to control levels. With [3H]Ins(1,4,5)P3 as a ligand, Scatchard analyses of the concentration-dependent saturation curves showed that the density of striatal intracellular Ins(1,4,5)P3 receptors was increased by 53%. The levels of SP and cellular Ins(1,4,5)P3, and the density of Ins(1,4,5)P3 receptors in the cerebellum, used as a positive control, were unchanged by chronic naltrexone treatment. The findings of opiate antagonist-induced increases in SP striatal content and Ins(1,4,5)P3 receptor densities, appear to support the concept of a role of endogenous opioids in the regulation of SP receptor activity. The data also suggest that inter-regulatory mechanisms exist between phospholipase C/phosphoinositide-coupled receptors such as SP receptors, and adenylate cyclase-coupled inhibitory receptors, such as opioid receptors.
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PMID:Regulation of substance P receptor system in rat striatum by chronic naltrexone treatment. 750 77

Src homology 2 (SH2) domains provide specificity to intracellular signaling by binding to specific phosphotyrosine (phospho-Tyr)-containing sequences. We recently developed a technique using a degenerate phosphopeptide library to predict the specificity of individual SH2 domains (src family members, Abl, Nck, Sem5, phospholipase C-gamma, p85 subunit of phosphatidylinositol-3-kinase, and SHPTP2 (Z. Songyang, S. E. Shoelson, M. Chaudhuri, G. Gish, T. Pawson, W. G. Haser, F. King, T. Roberts, S. Ratnofsky, R. J. Lechleider, B. G. Neel, R. B. Birge, J. E. Fajardo, M. M. Chou, H. Hanafusa, B. Schaffhausen, and L. C. Cantley, Cell 72:767-778, 1993). We report here the optimal recognition motifs for SH2 domains from GRB-2, Drk, Csk, Vav, fps/fes, SHC, Syk (carboxy-terminal SH2), 3BP2, and HCP (amino-terminal SH2 domain, also called PTP1C and SHPTP1). As predicted, SH2 domains from proteins that fall into group I on the basis of a Phe or Tyr at the beta D5 position (GRB-2, 3BP2, Csk, fps/fes, Syk C-terminal SH2) select phosphopeptides with the general motif phospho-Tyr-hydrophilic (residue)-hydrophilic (residue)-hydrophobic (residue). The SH2 domains of SHC and HCP (group III proteins with Ile, Leu, of Cys at the beta D5 position) selected the general motif phospho-Tyr-hydrophobic-Xxx-hydrophobic, also as predicted. Vav, which has a Thr at the beta D5 position, selected phospho-Tyr-Met-Glu-Pro as the optimal motif. Each SH2 domain selected a unique optimal motif distinct from motifs previously determined for other SH2 domains. These motifs are used to predict potential sites in signaling proteins for interaction with specific SH2 domain-containing proteins. The Syk SH2 domain is predicted to bind to Tyr-hydrophilic-hydrophilic-Leu/Ile motifs like those repeated at 10-residue intervals in T- and B-cell receptor-associated proteins. SHC is predicted to bind to a subgroup og these same motifs. A structural basis for the association of Csk with Src family members is also suggested from these studies.
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PMID:Specific motifs recognized by the SH2 domains of Csk, 3BP2, fps/fes, GRB-2, HCP, SHC, Syk, and Vav. 751 Dec 10


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