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 role of phospholipids in the activity of UDP-D-galactose: D-xylose galactosyltransferase (galactosyltransferase I) from embryonic chick cartilage was investigated. Phospholipase C treatment of particulate galactosyltransferase I caused inactivation of this enzyme to the extent of 60 to 70% as well as hydrolysis of 75 to 80% of the membrane phospholipids. Addition of phospholipid restored activity to nearly control levels. The order of effectiveness of various phopholipids in reactivating phospholipase C-treated galactosyltransferase I was as follows: lysophosphatidylcholine, lysophosphatidylethanolamine, phosphatidylethanolamine, phosphatidylcholine. The effect of phospholipase A on galactosyltransferase I activity was also examined and was found to be concentration-dependent. At concentrations less than 10 mug/mg of pellet protein, phospholipase A slightly activated galactosyltransferase I. whereas at higher concentrations it inhibited the activity in a manner similar to phospholipase C. Galactosyltransferase I was activated moderately and also solubilized by treatment with Nonidet P-40 in the presence of 0.5 M KCl. Following solubilization and purification by gel filtration and affinity chromatography, galactosyltransferase I could be inactivated by detergent removal by dialysis and subsequently reactivated by addition of detergent. Neither phospholipase C treatment nor exogenous phospholipid had any significant effect on three of the other chondroitin sulfate glycosyltransferases (UDP-D-xylose: core protein xylosyltransferase, UDP-D-glucuronic acid:3-O-beta-D-galactosyl-D-galactose glucuronosyltransferase, and UDP-N-acetyl-D-galactosamine:(BlcUA-GalNAc-4-sulfate)j N-acetylgalactosaminyltransferase). On lipid analysis by thin layer chromatography, phosphatidylcholine and phosphatidylethanolamine were found to be the major phospholipids of particulate and solubilized glycosyltransferase preparations from embryonic chick cartilage, while lysopholipids of particulate and solubilized glycosyltransferase preparations from embryonic chick cartilage, while lysophosphatidylcholine and lysophosphatidylethanolamine were barely detectable components. The concentration of these specific phospholipids was diminished greatly following phospholipase C treatment.
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PMID:Biosynthesis of chondroitin sulfate. Role of phospholipids in the activity of UDP-D-galactose: D-xylose galactosyltransferase. 94 16

A staphylococcal exotoxin that causes epidermolysis when injected into the skin of the newborn mouse and man was highly purified by coventional biochemical techniques. With Staphylococcus aureus EV, the epidermolytic toxin was a major protein component of supernatant culture fluids. The initial step in purification was zone electrophoresis in Pevikon carried out at pH 9.0, the isoelectric point of alpha-hemolytic toxin, which remained near the origin. Fractions containing the epidermolytic toxin, but free of alpha-toxin, were then subjected to cation exchange chromatography on carboxymethyl-Sephadex C-50 to remove trace contaminants. A major highly purified epidermolytic toxin migrated as a single band in polyacrylamide gel electrophoresis, sedimented as a single component in the analytical ultracentrifuge, and elicited a single precipitating antibody after injection into rabbits. A smaller amount of a second epidermolytic toxin, identical in molecular weight and antigenicity but differing in electrophoretic behavior from the major molecular species, was also identified. The epidermolytic factor had a molecular weight of 28,600 +/- 400 by sodium dodecyl sulfate-acrylamide electrophoresis and 32,500 +/- 120 by approach to sedimentation equilibrium.
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PMID:Purification and characterization of a staphylococcal epidermolytic toxin. 126 65

Human umbilical vein endothelial cells (HUVEC) were found by Western blot analysis to express three membrane-bound C regulatory proteins, decay-accelerating factor (DAF), membrane cofactor protein (MCP) and CD59. DAF was detected on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a 70-kDa molecule under nonreducing conditions in 2% deoxycholate extracts of HUVEC, MCP as a 63-kDa protein and CD59 as a 20-kDa molecule. Northern blot analysis revealed the presence of two species of mRNA expressed in HUVEC, which hybridized to a cDNA probe specific for DAF, with sizes of about 2.0 kb and 2.7 kb. MCP mRNA was detected at 4.2 kb and a CD59 cDNA probe hybridized with three mRNA species with sizes of about 800, 1400 and 2000 bp. DAF and CD59 were released from the surface of HUVEC by phosphatidylinositol-phospholipase C, demonstrating that both are attached to the cell membrane by means of a glycolipid anchor. The relative contribution of DAF, MCP and CD59 in regulating the sensitivity to lysis of HUVEC by autologous complement was determined by incubation of sensitized endothelial cells with F(ab')2 fragments of polyclonal antibodies raised against these proteins. The susceptibility of sensitized cells to lysis by homologous complement was markedly increased in the presence of F(ab')2 anti-CD59 and to a lesser, but significant, extent in the presence of F(ab')2 anti-DAF. F(ab')2 anti-MCP did not significantly alter the susceptibility of HUVEC to complement-mediated lysis.
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PMID:Relative roles of decay-accelerating factor, membrane cofactor protein, and CD59 in the protection of human endothelial cells against complement-mediated lysis. 128 Feb 24

The effective resolution of human platelet cytosolic phosphoinositide-phospholipase C (PLC) revealed five distinct activity peaks by Q-Sepharose and heparin-Sepharose column chromatographies when assayed using phosphatidylinositol (PI) and phosphatidylinositol 4,5-bisphosphate (PIP2). The results of Western blotting analysis with various antibodies against PLC isozymes showed that peak-Ia (PLC-delta type), peak-Ib (PLC-gamma 1 type), and peak-IIc (PLC-beta type) and two unidentified activity peaks (PLC-IIa and PLC-IIb) were present in human platelet cytosol. A protein with guanosine 5'-3-O-(thio)triphosphate-binding activity was coeluted with the PLC-IIa and was purified to homogeneity. It exhibited 86- and 42-kDa polypeptide bands upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis which were identified as gelsolin and actin by immunostaining, respectively. Large amounts of gelsolin/actin (1:1) complex "gelsolin complex" were detected in the PLC-delta and PLC-gamma 1 fractions. The PLC-gamma 1 and the gelsolin complex were co-immunoprecipitated by the antibody raised against PLC-gamma 1. Furthermore, the partially purified bovine brain PLC-gamma 1 fraction also was found to be associated with the gelsolin complex and the association was released by the addition of 1% sodium cholate. This finding has prompted us to examine effects of the gelsolin complex and the free gelsolin on activities of the above PLC isoforms from platelet cytosol. The gelsolin complex did not affect the PIP2 hydrolyzing activities of all PLC isoforms. In contrast, the purified gelsolin inhibited distinctly PIP2 hydrolyses by PLC-Ia (delta), PLC-Ib (gamma 1), and PLC-IIa (unidentified), whereas the inhibitory effects for PLC-IIb (unidentified) and PLC-IIc (beta) were moderate. The inhibitory effect of gelsolin on PIP2-hydrolysis by PLC-gamma 1 was diminished by a large amount of PIP2 substrate. These results suggested that the inhibition of PLC by gelsolin is due to sequestration of substrate PIP2 by its competitive binding.
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PMID:Effects of gelsolin on human platelet cytosolic phosphoinositide-phospholipase C isozymes. 131 7

Heparan sulfate proteoglycans (HSPG) are ubiquitous constituents of mammalian cell surfaces and most extracellular matrices. A portion of the cell surface HSPG is anchored via a covalently linked glycosyl-phosphatidylinositol (Pl) residue, which can be released by treatment with a glycosyl-Pl specific phospholipase C (Pl-PLC). We report that exposure of bovine aortic endothelial and smooth muscle cells to Pl-PLC resulted in release of cell surface-associated, growth-promoting activity that was neutralized by antibasic fibroblast growth factor (bFGF) antibodies. Active bFGF was also released by treating the cells with bacterial heparitinase. Under the same conditions there was no release of mitogenic activity from cells (BHK-21, NIH/3T3, PF-HR9) that expressed little or no bFGF, as opposed to Pl-PLC-mediated release of active bFGF from the same cells transfected with the bFGF gene. The released bFGF competed with recombinant bFGF in a radioreceptor assay. Addition of Pl-PLC to sparsely seeded vascular endothelial cells resulted in a marked stimulation of cell proliferation, but there was no mitogenic effect of Pl-PLC on 3T3 fibroblasts. Studies with exogenously added 125I-bFGF revealed that about 6.5% and 20% of the cell surface-bound bFGF were released by treatment with Pl-PLC and heparitinase, respectively. Both enzymes also released sulfate-labeled heparan sulfate from metabolically labeled 3T3 fibroblasts. Pl-PLC failed to release 125I-bFGF from the subendothelial extracellular matrix (ECM), as compared to release of 60% of the ECM-bound bFGF by heparitinase. Our results indicate that 3-8% of the total cellular content of bFGF is associated with glycosyl-Pl anchored cell surface HSPG. This FGF may exert both autocrine and paracrine effects, provided that it is released by Pl-PLC and adequately presented to high affinity bFGF cell surface receptor sites.
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PMID:Release of cell surface-associated basic fibroblast growth factor by glycosylphosphatidylinositol-specific phospholipase C. 131 17

A 60-kDa protein homologous to phosphoinositide-specific phospholipase C-alpha was purified to apparent homogeneity on sodium dodecyl sulfate-polyacrylamide gel electrophoresis from the rough endoplasmic reticulum of rat liver through three sequential chromatographies on DEAE Toyopearl 650, AF-heparin Toyopearl 650M, and TSK gel G3000SW. The purified protein was monomeric, with an M(r) of 60,000. Eight types of protein were further separated from the 60-kDa protein and named ER60A-ER60H according to the order of their elution from a TSK gel DEAE-5PW column. They were essentially identical in terms of immunochemical properties and the NH2-terminal amino acid sequence. The partial amino acid sequence of ER60F showed homology to that of phosphoinositide-specific phospholipase C-alpha. ER60A-ER60H showed no phosphoinositide-specific phospholipase C activity. However, ER60A-ER60H catalyzed cleavage of themselves and the endoplasmic reticulum proteins protein disulfide-isomerase and calreticulin. Proteolytic degradation was inhibited by p-chloromercuribenzoate. These results indicate that ER60A-ER60H comprise a group of endoplasmic reticulum resident proteins and show thiol group-related proteolytic activity.
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PMID:Protein degradation by the phosphoinositide-specific phospholipase C-alpha family from rat liver endoplasmic reticulum. 132 29

The addition of ammonium sulfate to starved yeast cells leads to a 3- to 4-fold rapid increase of the second messengers inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG), the products of phosphoinositide-specific phospholipase C (PI-PLC). This response is reduced by dissecting the RAS-activating Cdc25 protein, and is completely abolished by the cdc25-1 mutation even at permissive temperature. Starved cdc25-1 mutant cells have a strongly reduced IP3 content, but an at least 10-fold increased DAG level compared to the isogenic wild-type strain. NH4 does not stimulate cAMP synthesis, and glucose does not induce IP3 and DAG. Our data suggest that the Cdc25 protein controls a nitrogen-specific signalling pathway involving the effector PI-PLC, in addition to the glucose-induced activation of adenylyl cyclase (AC).
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PMID:CDC25-dependent induction of inositol 1,4,5-trisphosphate and diacylglycerol in Saccharomyces cerevisiae by nitrogen. 132 32

Treating the liposome-intercalatable heparan sulfate proteoglycans from human lung fibroblasts and mammary epithelial cells with heparitinase and chondroitinase ABC revealed different core protein patterns in the two cell types. Lung fibroblasts expressed heparan sulfate proteoglycans with core proteins of approximately 35, 48/90 (fibroglycan), 64 (glypican), and 125 kDa and traces of a hybrid proteoglycan which carried both heparan sulfate and chondroitin sulfate chains. The mammary epithelial cells, in contrast, expressed large amounts of a hybrid proteoglycan and heparan sulfate proteoglycans with core proteins of approximately 35 and 64 kDa, but the fibroglycan and 125-kDa cores were not detectable in these cells. Phosphatidylinositol-specific phospholipase C and monoclonal antibody (mAb) S1 identified the 64-kDa core proteins as glypican, whereas mAb 2E9, which also reacted with proteoglycan from mouse mammary epithelial cells, tentatively identified the hybrid proteoglycans as syndecan. The expression of syndecan in lung fibroblasts was confirmed by amplifying syndecan cDNA sequences from fibroblastic mRNA extracts and demonstrating the cross-reactivity of the encoded recombinant core protein with mAb 2E9. Northern blots failed to detect a message for fibroglycan in the mammary epithelial cells and in several other epithelial cell lines tested, while confirming the expression of both glypican and syndecan in these cells. Confluent fibroblasts expressed higher levels of syndecan mRNA than exponentially growing fibroblasts, but these levels remained lower than observed in epithelial cells. These data formally identify one of the cell surface proteoglycans of human lung fibroblasts as syndecan and indicate that the expression of the cell surface proteoglycans varies in different cell types and under different culture conditions.
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PMID:Differential expression of cell surface heparan sulfate proteoglycans in human mammary epithelial cells and lung fibroblasts. 133 31

CD59 is a 18-20-kDa membrane glycoprotein that inhibits formation of the membrane attack complex of complement (C) on homologous cells. In the present study we analyzed the expression and function of CD59 on human endothelial cells. Immunohistochemical analysis of renal cortex demonstrated a predominant expression of CD59 on peritubular capillary endothelial cells and glomerular endothelial cells. Flow cytometry analysis showed that human umbilical vein endothelial cells (HUVEC) expressed CD59 and the fluorescence intensity was approximately four times that of peripheral blood lymphocytes. CD59 is detected on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a single 20-kDa molecule in 2% deoxycholate extracts of HUVEC. CD59 was released from the surface of HUVEC by phosphatidylinositol-specific phospholipase C, demonstrating that it is attached to the cell membrane by means of a glycolipid anchor. The functional activity of CD59 expressed on HUVEC was studied. Blocking of CD59 antigen with F(ab')2 fragments of polyclonal anti-CD59 enhanced markedly the susceptibility of HUVEC to C-mediated lysis. This effect was dependent on the amount of blocking antibodies added. Northern blot analysis revealed the presence of three species of mRNA expressed in HUVEC, which hybridized to a cDNA probe specific for CD59, with sizes of about 800, 1400 and 2000 bp. These findings suggest that CD59 may be important in protection of endothelial cells against C-mediated damage at local sites of inflammation, thereby maintaining the vascular integrity in vivo.
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PMID:CD59 expressed by human endothelial cells functions as a protective molecule against complement-mediated lysis. 137 60

A number of cell surface proteins have been shown to be anchored to the plasma membrane by a covalently attached glycoinositol phospholipid (GPL) in amide linkage to the C-terminus of the mature protein. We applied several criteria to establish that folate binding protein (FBP) in brush border membranes of rat kidney contains a GPL anchor. Brush border membranes were isolated and labeled with [3H]folate, and the complex of FBP and [3H]folate was shown to be released to the supernatant by incubation with purified bacterial phosphatidylinositol-specific phospholipase C (PIPLC) but not by incubation with a purified bacterial phosphatidylcholine-specific phospholipase C. The FBP-[3H]folate complex both in crude extracts and after FBP purification by ligand-directed affinity chromatography interacted with Triton X-114 micelles, and prior incubation with PIPLC prevented this detergent interaction. Individual residues characteristic of GPL anchors were found to be covalently associated with FBP following polyacrylamide gel electrophoresis in sodium dodecyl sulfate. These included glucosamine and ethanolamine, which were radiolabeled by reductive methylation and identified by chromatography on an amino acid analyzer, and inositol phosphate, which was inferred by Western blotting with an anti-CRD antisera. This antisera gave positive immunostaining only after FBP had been cleaved by PIPLC, a reliable diagnostic of a GPL anchor. The relationship between GPL-anchored FBP in biological membranes and soluble FBP in biological fluids also is discussed.
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PMID:Folate binding protein from kidney brush border membranes contains components characteristic of a glycoinositol phospholipid anchor. 137 26


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