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)

A large number of mammalian proteins are anchored to the cell membrane by a glycosylphosphatidylinositol (GPI) anchor. Biosynthetic intermediates of the GPI anchor have been identified in mammalian cells. The early GPI precursors are sensitive to phosphatidylinositol (PI)-specific phospholipase C (PLC). However, all of the later GPI precursors, which contain 1 or more mannose residues, are PI-PLC-resistant, suggesting that there is another unidentified precursor. Here, we report the identification of this missing link. This GPI precursor can only be labeled with glucosamine and inositol, and is resistant to PI-PLC but sensitive to GPI-phospholipase D. It accumulates in large quantity only in mutants which are defective in the addition of the first mannose residue to the elongating GPI core. Thus, fatty acylation of glucosaminylphosphatidylinositol, to render it PI-PLC-resistant, is an obligatory step in the biosynthesis of mammalian GPI anchor precursors.
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PMID:Identification of a missing link in glycosylphosphatidylinositol anchor biosynthesis in mammalian cells. 131 4

The identification of free glycoinositol phospholipids (GPIs) following biosynthetic labeling with [3H]glucosamine in cultured cells has been reported by several laboratories. We applied this procedure to two of the cell types used in these studies, H4IIE hepatoma cells and isolated hepatocytes, but were unable to detect a [3H]glucosamine-containing lipid that met any of the criteria for GPIs, including sensitivity to phosphatidylinositol-specific phospholipase C (PIPLC) or GPI-specific phospholipase D. Part of the difficulty in radiolabeling a GPI by this procedure was the rapid metabolic conversion of [3H]glucosamine to galactosamine and neutral or anionic derivatives. A PIPLC-sensitive radiolabeled lipid was detected only after 16 h of labeling. The water-soluble fragments released from this lipid by PIPLC corresponded largely to myo-inositol 1,2-cyclic phosphate and myo-inositol 1-phosphate, products expected from PIPLC cleavage of phosphatidylinositol or lyso-phosphatidylinositol. In an alternative approach that we introduce here, free GPIs in lipid extracts from rat liver plasma membranes were labeled by reductive radiomethylation. This procedure, which radiomethylates primary and secondary amines, has been shown to label a glucosamine residue adjacent to inositol in all GPIs characterized to date. The labeled extracts were fractionated by two-dimensional thin-layer chromatography, and a cluster of polar labeled lipids were assigned as GPIs based upon the following observations. 1) They were cleaved by PIPLC, 2) after hydrolysis in 6 N HCl, both radiomethylated glucosamine and a glucosamine-inositol conjugate were identified by cation exchange chromatography, and 3) hydrolysis in 4 M trifluoroacetic acid generated a fragment consistent with glucosamine-inositol-phosphate. These results illustrate new criteria for the identification of GPIs. The labeled GPIs also contained radiomethylated ethanolamine, another component found in GPI anchors of proteins and in mature lipid precursors of GPI anchors, suggesting that the liver plasma membrane GPIs retained considerable structural homology to GPI anchors.
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PMID:Identification of glycoinositol phospholipids in rat liver by reductive radiomethylation of amines but not in H4IIE hepatoma cells or isolated hepatocytes by biosynthetic labeling with glucosamine. 132 29

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

Inositol glycans were prepared from reductively radiomethylated human erythrocyte acetylcholinesterase by sequential treatment with Proteinase K, methanolic KOH, and phosphatidylinositol-specific phospholipase C. Four glycans denoted alpha-delta were resolved by anion exchange high performance liquid chromatography (HPLC). Each glycan was subjected to hydrolysis in 4 M trifluoroacetic acid, and their hexose and hexose phosphate compositions were determined by anion exchange HPLC. The predominant glycan alpha showed a relative stoichiometry of 2 mannoses, 1 mannose 6-phosphate, 1 radiomethylated glucosamine, 1 radiomethylated ethanolamine, and 1 inositol. In contrast, the stoichiometry of glycan beta was 1 mannose, 2 mannose 6-phosphates, 1 radiomethylated glucosamine, 2 radiomethylated ethanolamines, and 1 inositol. Glycans alpha and beta were analyzed by electrospray ionization-mass spectrometry, and respective parent ions of m/z 1266 and 1417 were observed. The fragmentation pattern produced by collision-induced dissociation mass spectrometry of these parent ions was consistent with a common linear core glycan sequence prior to radiomethylation of ethanolamine-phosphate-mannose - mannose - mannose - glucosamine - inositol. Glycan alpha contained a single additional radiomethylated phosphoethanolamine branching from the mannose adjacent to glucosamine, whereas glycan beta contained two additional radiomethylated phosphoethanolamines, one branching from each of the mannoses nearest to glucosamine. Trifluoroacetic acid hydrolysis did not cleave within the N,N-dimethylglucosamine-inositol-phosphate moiety in these glycans, and this component was resolved by anion exchange HPLC and structurally confirmed by mass spectrometry. Dephosphorylation of this component by treatment with 50% HF produced N,N-dimethylglucosamine-inositol, and this conjugate was shown to have a characteristic elution time on cation exchange chromatography in an amino acid analyzer. Both of these fragments involving an intact radiomethylated glucosamine-inositol bond are proposed as new diagnostic indicators in the search for minor glycoinositol phospholipids in cells and tissues.
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PMID:Glycan components in the glycoinositol phospholipid anchor of human erythrocyte acetylcholinesterase. Novel fragments produced by trifluoroacetic acid. 138 56

The present report describes the radioactive labeling of polar lipids in in vitro cultures of Plasmodium falciparum as well as their extraction with organic solvents and their partial characterization by chemical and enzymatic methods. All substances detected could be cleaved by alkali, suggesting that they were esters rather than sphingolipids or compounds containing alkyl groups. Dolichol-cycle intermediates were not detected. Phosphatidylinositol, phosphatidylethanolamine, and phosphatidylcholine were labeled by fatty acids and inositol or ethanolamine, respectively, confirming their de novo synthesis by the parasite. Metabolic labeling with glucosamine and cleavage by phosphatidylinositol-specific phospholipase C provided evidence of the formation of N-acetyl-glucosaminyl-phosphatidylinositol, an obligate precursor in the biosynthesis of glycosylphosphatidylinositol membrane anchors of proteins.
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PMID:Labeling and initial characterization of polar lipids in cultures of Plasmodium falciparum. 149 20

Four major glycolipids were extracted from Toxoplasma gondii tachyzoites which were metabolically labeled with tritiated glucosamine, mannose, palmitic and myristic acid, ethanolamine, and inositol. Judging from their sensitivity to a set of enzymatic and chemical tests, these glycolipids share the following properties with the glycolipid moiety of the glycosylphosphatidylinositol anchor (GPI anchor) of the major surface protein, P30, of T. gondii: 1) a nonacetylated glucosamine-inositol phosphate linkage; 2) sensitivity toward phosphatidylinositol-specific phospholipase C and nitrous acid; 3) identity of HF-dephosphorylated GPI glycan backbone between three glycolipids and the HF-dephosphorylated core glycan of the GPI anchor of the major surface protein P30; 4) the presence of a linear core glycan structure blocked by an ethanolamine phosphate residue(s). Taken together with the nature of radiolabeled precursors incorporated into these glycolipids, the data indicate that these GPIs are involved in the biosynthesis of the GPI-membrane anchors of T. gondii.
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PMID:A family of glycolipids from Toxoplasma gondii. Identification of candidate glycolipid precursor(s) for Toxoplasma gondii glycosylphosphatidylinositol membrane anchors. 153 1

The heparan sulphate (HS) proteoglycans associated with the cell layer of a rat osteosarcoma cell line [UMR 106-01 (BSP)] were compared with similar cell-associated proteoglycans from other cells, and their interaction with the plasma membrane was studied. HS proteoglycans were metabolically labelled by incubation of cell cultures with [3H]glucosamine or [3H]leucine and [35S]sulphate. HS proteoglycan core protein preparation generated by heparitinase digestion of the major species from UMR 106-01 (BSP) cells co-migrated on PAGE with identical preparations from ovarian granulosa cells and parathyroid cells (at approximately 70 kDa). The hydrophobic nature of the major HS proteoglycans from these diverse cell lines, based on elution position from octyl-Sepharose, were also comparable. Linkages of the HS proteoglycan to the cell membrane were investigated by labelling plasma-membrane preparations with a lipid soluble photoactivatable reagent, 3-(trifluoromethyl)-3- (m-[125I]iodophenyl)diazirine (TID), which selectively labels plasma-membrane-spanning peptide domains. Purified HS proteoglycan from UMR 106-01 (BSP) cells was shown to be accessible to the [125I]TID, and the core protein portion of the molecule was labelled, confirming its close association with the plasma membrane. Approx. 36% of 35S-labelled HS proteoglycans were released from the cell surface by phospholipase C (Bacillus thuringiensis), which specifically cleaves phosphatidylinositol-linked proteins. In the presence of insulin, the metabolism of the phospholipase C-sensitive population was unaltered; however, release of the phospholipase C-insensitive population into the medium was increased. These data indicate that a subpopulation of HS proteoglycans are covalently bound to the plasma membrane by a glycosylphosphatidylinositol structure, with the remainder representing those species directly inserted into the plasma membrane via a hydrophobic peptide domain. These observations are similar to those reported for ovarian granulosa cells [Yanagishita & McQuillan (1989) J. Biol. Chem. 264 17551-17558], and thus may represent a general phenomenon for many cell types.
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PMID:Plasma-membrane-intercalated heparan sulphate proteoglycans in an osteogenic cell line (UMR 106-01 BSP). 163 8

Ryals et al. (Ryals, P.E., Pak, Y., and Thompson, G. A., Jr., (1991) J. Biol. Chem. 266, 15048-15053) have described and partially characterized phosphatidylinositol glycans (PI glycans) present in Tetrahymena mimbres. We now describe the time course of PI glycan labeling from exogenous [3H]myristate, [14C]glucosamine, and [3H]ethanolamine. Over the first 2-12 h following pulse radioisotope addition a sizeable proportion of the radioactivity associated with the protein pellet remaining after cell delipidation existed as PI glycans. These compounds were distributed throughout the cell, with the largest proportion at 12 h being associated with a fraction containing mitochondria, lysosomes, and peroxisomes. However, by 24 h radioactivity had nearly disappeared from the PI glycans and had become associated with proteins by a process that was almost totally inhibited by cycloheximide or tunicamycin. PI glycans appeared to be incorporated mainly into a protein migrating on sodium dodecyl sulfate-polyacrylamide gel electrophoresis in a relatively broad band with an apparent molecular mass of 24-29 kDa. The exact mobility of the protein band within this molecular weight range was dependent upon the growth temperature of the cells. The apparent molecular masses of the principal PI-anchored proteins formed by other closely related Tetrahymena species varied widely, ranging from 22 to 76 kDa. The PI-anchored proteins may belong to a group of surface proteins known as immobilization antigens. Treatment of 24-h-labeled T. mimbres cells with phosphatidylinositol-specific phospholipase C in vivo released labeled proteins from the cells. Some labeled proteins were present even in the medium of control, non-phospholipase-treated cells. Tetrahymena PI glycans appear to accumulate in a metabolic pool from which they are gradually removed for attachment to externally oriented PI-anchored proteins. Tetrahymena is a versatile system well suited for studying the regulation of PI-anchored protein biochemistry.
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PMID:Phosphatidylinositol glycan formation and utilization by the ciliate Tetrahymena mimbres. 165 18

Insulin action is thought to be mediated by an inositol-, glucosamine- and galactose-containing oligosaccharide liberated by phosphodiesterase hydrolysis of a glycosyl-phosphatidylinositol. This oligosaccharide inhibits insulin biosynthesis and secretion in pancreatic islets. In the present study, two main glycolipids (peak I and II) were resolved by sequential TLC of lipids extracted from islet cells labelled with tritiated glucosamine, galactose or myristate. The two glycolipids displayed comparable sensitivity to beta-galactosidase but differed from one another by their sensitivity to phosphatidylinositol-specific phospholipase C. Moreover, structural heterogeneity within each peak was suggested by their partial resistance to nitrous acid deamination. These findings support the presence in islet cells of glycolipids similar to those currently considered as a possible postreceptor target for insulin in other cell types.
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PMID:Metabolic labelling and partial characterization of glycophospholipids in pancreatic islet cells. 165 34

Species-specific monoclonal antibodies to Leishmania tropica, T11 and T13-15, recognize membranal and secreted antigens. The membrane form of the antigen migrates on sodium dodecyl sulfate-polyacrylamide gels with a diffuse molecular weight from 15 to 50 kDa and can be labeled with palmitic acid, myoinositol, galactose, glucosamine, and inorganic phosphate. Both phosphate and sugar-labeled material were isolated from metabolically labeled promastigotes by affinity chromatography on antibodies coupled to Sepharose 4B. No binding to Ricinus communis agglutinin was observed. This material behaves like lipophosphoglycans from other Leishmania but contains unique species-specific epitopes. It is susceptible to cleavage by phospholipase C and after digestion no longer partitions into the detergent phase following a Triton X-114 extraction. All four monoclonal antibodies appear to recognize a carbohydrate epitope on the lipophosphoglycan since periodate treatment of this material bound to nitrocellulose essentially eliminated antibody binding. In addition, T15 binding could be blocked by 5 mM mannose-6-PO4 and fructose-1- or 6-PO4, but not by mannose, glucose, fructose, or the additional PO4 derivatives examined. The antibodies recognize a similar but not identical epitope, as demonstrated by a competitive radioimmunoassay using 125I-labeled T11, T13, and T15. Expression of surface antigen is elevated during the promastigote stationary phase.
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PMID:Leishmania tropica: characterization of a lipophosphoglycan-like antigen recognized by species-specific monoclonal antibodies. 168 34


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