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)

Membranous and soluble forms of rat liver alkaline phosphatase were selectively prepared by extracting microsomes with n-butanol at pH 8.5 and 5.5, respectively, and purified in homogeneous forms by the method previously established (Miki et al. (1986) Eur. J. Biochem. 160, 41-48). When subjected to polyacrylamide gel electrophoresis, the two forms migrated to the same position in the presence of sodium dodecyl sulfate, while the membranous form remained at the top of gels in the absence of the detergent. Treatment of the membranous form with phosphatidylinositol-specific phospholipase C resulted in its conversion to a soluble form with the same electrophoretic mobility even in the absence of the detergent as that of the soluble form extracted at pH 5.5. Automated Edman degradation analysis showed that the two forms have the same N-terminal amino acid sequence up to the 30th residue determined. Chemical analyses of hydrolysates of the two forms by gas-liquid chromatography demonstrated that the membranous form contains palmitic acid, stearic acid, and inositol, while the soluble form contains inositol but is devoid of the fatty acids. Taken together, these results suggest that rat liver alkaline phosphatase is covalently attached to phosphatidylinositol acylated with palmitic acid and stearic acid, which functions as the membrane-anchoring domain of the enzyme molecule.
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PMID:Chemical identification of lipid components in the membranous form of rat liver alkaline phosphatase. 283 51

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

The N and C terminals and tyrosine-phosphorylating site of the middle-sized tumor antigen of polyoma virus were chemically synthesized. The sequences of these peptides were Met-Asp-Arg-Val-Leu-Ser-Arg-Ala-Asp-Lys (N-MT), Met-Leu-Phe-Ile-Leu-Ile-Lys-Arg-Ser-Arg-His-Phe (C-MT), and Glu-Glu-Glu-Glu-Tyr-Met-Pro-Met-Glu (MT-Tyr), respectively. Among these peptides, the C-MT peptide inhibited phospholipase A2 (EC 3.1.1.4), phospholipase C (EC 3.1.4.3), and phospholipase D (EC 3.1.4.4). In addition, phosphatidylinositol-specific phospholipase C (EC 3.1.4.10) was also inhibited by this peptide. To study the mechanism of the inhibition, kinetic analysis was performed using phospholipase A2 from porcine pancreas. The degree of inhibition of phospholipase was dose dependent, and maximal inhibition was observed at pH 8.8. This peptide inhibited phospholipase A2 in a competitive manner for low-affinity sites of Ca2+, and in a noncompetitive manner for phospholipid substrates. When a fatty acid in the 2 position of the glycerol moiety of phosphatidylcholine was replaced by palmitic acid (C16:0), oleic acid (C18:1), linoleic acid (C18:2), eicosatrienoic acid (C20:3), or arachidonic acid (C20:4), the degree of inhibition of phosphatidylcholine hydrolysis by the C-MT peptide decreased. Inhibition of phospholipase A2 by the C-MT peptide was reversed by low concentrations of sodium deoxycholate but not by Triton X-100 or Nonidet P40, nonionic detergents. These detergents and the modification of acyl groups altered the micellar state of phospholipids. These results, taken together, suggest that the binding of the C-MT peptide near the low-affinity Ca2+ binding sites modifies the interaction of phospholipid substrates with the active center of phospholipase A2.
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PMID:Inhibition of phospholipases by Met-Leu-Phe-Ile-Leu-Ile-Lys-Arg-Ser-Arg-His-Phe, C terminus of middle-sized tumor antigen. 285 79

Recent evidence shows that the mature Thy-1 surface glycoprotein lacks the C-terminal amino acids 113 to 143 predicted from the cDNA sequence and is anchored in the plasma membrane by a complex, phosphatidylinositol-containing glycolipid attached to the alpha-carboxyl group of amino acid 112. Here we studied the biosynthesis of Thy-1 in two previously described and two newly isolated Thy-1-deficient mutant cell lines. Somatic cell hybridization indicated that their mutations affected some processing step rather than the Thy-1 structural gene. The Thy-1 made by mutants of classes C, F, and H bound detergent but, in contrast to wild-type Thy-1, their detergent-binding moieties could not be removed by phospholipase C. In addition, tryptophan, which only occurs in position 124, was incorporated into Thy-1 of these mutants but not of wild-type cells. Last, the Thy-1 of wild-type but not mutant cells could be radiolabeled with [3H]palmitic acid. Together, these findings strongly suggest that mutants of classes C, F, and H accumulate a biosynthetic intermediate of Thy-1 which retains at least part of the hydrophobic C-terminal peptide. The Thy-1 of these mutants remained endoglycosidase H sensitive, suggesting that it accumulated in the rough endoplasmic reticulum or the Cis-Golgi. A different Thy-1 intermediate was found in a class B mutant cell line: the Thy-1 of this mutant was 2 kilodaltons smaller than the Thy-1 of other cell lines, did not bind detergent, and was rapidly secreted via a normal secretory pathway.
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PMID:No glycolipid anchors are added to Thy-1 glycoprotein in Thy-1-negative mutant thymoma cells of four different complementation classes. 289 21

P30, the major surface antigen of the parasitic protozoan Toxoplasma gondii, can be specifically labeled with [3H]palmitic acid and with myo-[2-3H]inositol. The fatty acid label can be released by treatment of P30 with phosphatidylinositol-specific phospholipase C (PI-PLC). Such treatment exposes an immunological "cross-reacting determinant" first described on Trypanosoma brucei variant surface glycoprotein. PI-PLC cleavage of intact parasites metabolically labeled with [35S]methionine results in the release of intact P30 polypeptide in a form which migrates faster in polyacrylamide gel electrophoresis. These results argue that P30 is anchored by a glycolipid. Results from thin layer chromatography analysis of purified [3H] palmitate-labeled P30 treated with PI-PLC, together with susceptibility to mild alkali hydrolysis and to cleavage with phospholipase A2, suggest that the glycolipid anchor of T. gondii P30 includes a 1,2-diacylglycerol moiety.
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PMID:The major surface antigen, P30, of Toxoplasma gondii is anchored by a glycolipid. 292 21

Previous investigations in this laboratory have indicated that arachidonic acid stimulates a rapid, dose-dependent, and reversible increase in human placental lactogen (hPL) release which is not dependent on cyclooxygenase or lipoxygenase metabolism. To investigate further the mechanism by which arachidonic acid stimulates the release of hPL, the effects of arachidonic acid on phosphoinositide hydrolysis were examined in an enriched cell culture population of term human syncytiotrophoblast. Phosphoinositide hydrolysis was assayed by three methods: the release of 3H from perfused cells prelabeled with [3H]myoinositol, the measurement of inositol phosphate accumulation, and the distribution of radioactivity in phospholipids separated by two-dimensional thin layer chromatography after exposure of 32P-labeled placental cells to arachidonic acid. Arachidonic acid stimulated a concentration-dependent, rapid, and reversible increase in the release of both [3H]myoinositol and hPL from perfused placental cells. This effect was not inhibited by prior incubation of cells with indomethacin (20 microM). In contrast, palmitic acid and oleic acid stimulated phosphoinositide hydrolysis only at a high concentration (100 microM). Arachidonic acid also stimulated the rapid appearance of inositol monophosphate in placental cells. The effect of arachidonic acid was specific for hydrolysis of phosphoinositides and phosphatidylserine and did not involve other phospholipids. Since phosphoinositide hydrolysis is associated with hormone release in a variety of secretory systems, these results suggest that the stimulation of hPL release by arachidonic acid may be mediated, at least in part, by the activation of phospholipase C.
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PMID:Arachidonic acid stimulates phosphoinositide hydrolysis and human placental lactogen release in an enriched fraction of placental cells. 300 98

Cultured endothelial cells from human umbilical vein were incubated for 20 h at 37 degrees C in the presence of [U-14C]arachidonic acid. Around 60-70% of the radioactive fatty acid was incorporated into cell lipids and was predominantly found in phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol and triacylglycerol (39%, 33%, 13% and 6.5% of total incorporated radioactivity, respectively). Stimulation of the cells with human thrombin (2 U/ml) or calcium ionophore A23187 (5 microM) promoted the release into supernatants of arachidonic acid, 6-ketoprostaglandin F1 alpha, prostaglandins E2 and F2 alpha, in decreasing order of importance. The amount of secreted material was 4-fold higher with A23187, compared to thrombin. Parallel to the liberation process, phosphatidylcholine underwent a rapid decrease of radioactivity with both agonists, suggesting the involvement of a Ca2+-dependent phospholipase A2. Phosphatidylethanolamine displayed a minor decrease with A23187, whereas some reacylation was observed at 10 min with thrombin. Phosphatidylinositol was non-significantly affected in thrombin-stimulated cells, whereas A23187 promoted an early but minor decrease, followed by resynthesis. In contrast to A23187, thrombin was also able to promote a significant hydrolysis of triacylglycerol, which might thus be implicated in the process of arachidonate liberation. Finally, radioactive phosphatidic acid and diacylglycerol appeared in endothelial cells, in response to the two agonists. However, diacylglycerol formation did not parallel that of phosphatidic acid, especially with A23187. Determination of the 14C/3H ratio of the different lipids upon cell labelling with both [14C]arachidonic acid and [3H]palmitic acid revealed that diacylglycerol and phosphatidic acid are hardly derived from inositol-phospholipid breakdown by phospholipase C. Other possible pathways involving for instance phospholipase C splitting of phosphatidylcholine are discussed.
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PMID:Pathways of arachidonic acid liberation in thrombin and calcium ionophore A23187-stimulated human endothelial cells: respective roles of phospholipids and triacylglycerol and evidence for diacylglycerol generation from phosphatidylcholine. 309 49

Phosphatidylinositol anchors human placental-type alkaline phosphatase (PLAP) to both syncytiotrophoblast and tumour cell plasma membranes. PLAP activity was released from isolated human placental syncytiotrophoblast plasma membranes and the surface of tumour cells with a phospholipase C from Bacillus cereus. This was a specific event, not the result of proteolysis or membrane perturbation, but the action of a phosphatidylinositol-specific phospholipase C in the preparation. Soluble PLAP, released with B. cereus phospholipase C and purified by immunoaffinity chromatography, ran on SDS-PAGE as a 66-kDa band. This corresponded to intact PLAP molecules. The protease bromelain cleaved lower-molecular-mass PLAP (64 kDa) from the membranes. Flow cytometry demonstrated that B. cereus phospholipase C released human tumour cell membrane PLAP in preference to other cell-surface molecules. This was in contrast to the non-specific proteolytic action of bromelain or Clostridium perfringens phospholipase C, which had no effect on membrane PLAP expression. Radiolabelling of tumour cells with fatty acids indicated PLAP to be labelled with both [3H]myristic and [3H]palmitic acid. This fatty-acid--PLAP bond was sensitive to pH 10 hydroxylamine treatment indicating an O-ester linkage.
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PMID:Attachment of human placental-type alkaline phosphatase via phosphatidylinositol to syncytiotrophoblast and tumour cell plasma membranes. 312 11

The biosynthesis and post-translational modification of placental alkaline phosphatase were studied in human choriocarcinoma cells, JEG-3. Pulse-chase experiments with [35S]methionine demonstrated that placental alkaline phosphatase was synthesized as a major precursor form with Mr 63,000, which was then converted to a mature form with Mr 66,000, by processing of its N-linked oligosaccharides from the high-mannose type to the complex type. In addition, the two forms of the protein were found to be modified by a glycophospholipid, components of which were characterized by metabolic incorporation into placental alkaline phosphatase of 3H-labeled compounds such as myo-inositol, palmitic acid, stearic acid, mannose, glucosamine, and ethanolamine. When placental alkaline phosphatase labeled with these compounds was treated with phosphatidylinositol-specific phospholipase C or papain, the phospholipase C removed only the 3H-labeled fatty acids, whereas papain, that is known to cleave the C-terminal region, released all the radioactive glycolipid components including [3H]ethanolamine. More detailed analysis with shorter pulse-chase experiments demonstrated that placental alkaline phosphatase was primarily synthesized as a form with Mr 64,500 which was not yet labeled with [3H]palmitic acid. This form was converted by papain digestion to the above-mentioned major precursor with Mr 63,000. Taken together, these results suggest that placental alkaline phosphatase is initially synthesized as the precursor with Mr 64,500, which is immediately converted to the intermediate form with Mr 63,000 by simultaneously occurring proteolysis of the C terminus and replacement by the glycophospholipid, and finally to the mature form with Mr 66,000 by terminal glycosylation of its N-linked oligosaccharides. The glycophospholipid thus attached is considered to function as the membrane-anchoring domain of placental alkaline phosphatase.
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PMID:Biosynthesis of placental alkaline phosphatase and its post-translational modification by glycophospholipid for membrane-anchoring. 334 38

Receptor-mediated activation of many cells, including blood platelets, leads to changes at the cytoplasmic side of the membrane. In platelets, phospholipases, such as phospholipase C and phospholipase A2, have been shown to become activated. From phospholipids they generate the second messengers diacylglycerol and inositol phosphate(s) and fatty acids, respectively. At the same time, actin polymerization and reorganization of actin filaments into bundles and networks occurs. Here, the association of lipids, radiolabeled either with saturated (palmitic acid) or unsaturated (arachidonic acid) fatty acids, with the cytoskeletons of resting and activated human blood platelets was studied. The relative binding of lipid components to the cytoskeleton of activated platelets labeled with palmitic acid is six times higher than that of platelets labeled with arachidonic acid. Analysis of lipids associated with isolated cytoskeletons of resting and activated platelets (labeled with palmitic acid) showed a 30-fold increase in the binding of labeled lipids to the cytoskeletal structures during activation. Both diacylglycerol and fatty acids were found to be associated with the cytoskeleton of activated platelets. Gel filtration, chromatofocusing, and immunoprecipitation studies demonstrated tight binding of these lipids to alpha-actinin. alpha-Actinin is one of the proteins that rapidly becomes associated with the cytoskeleton during platelet aggregation; it is also one of the molecules proposed to act as an actin-membrane linker. The results reported indicate a possible participation of alpha-actinin, fatty acids, and the phosphoinositide-derived second messenger diacylglycerol in the regulation of cytoskeleton-membrane interactions. Together with the results of others they suggest a possible involvement of the phosphatidylinositol cycle in the assembly of actin filaments and their anchoring to membranes.
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PMID:Phosphatidylinositol cycle and its possible involvement in the regulation of cytoskeleton-membrane interactions. 334 87


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