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 lipophosphoglycan (LPG)-like glycoconjugate expressed on the cell surface of Trypanosoma cruzi epimastigotes was isolated, purified, and partially characterized. The glycoconjugate migrated as a homogeneous band (42 kDa) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Matrix-assisted laser desorption ionization mass spectral analysis of the native molecule indicated the presence of two major components whose molecular masses were about 18.4 and 22.5 kDa. The LPG could be metabolically labeled with [3H]galactose, [3H]mannose, [14C]glucose, or [3H]palmitic acid. Monosaccharide compositional analysis of the LPG indicated that galactose, glucosamine, and sialic acid predominate over mannose, galactosamine, and inositol. A peptide associated with the LPG molecule contained about 40 amino acid residues per inositol and had threonine as the predominant amino acid. The LPG showed strong binding to Ricinus communis agglutinin-1 and Tritium vulgare wheat germ agglutinin, indicating the presence of terminal beta 1,4-linked galactosyl residue(s) and N-acetylglucosamine, respectively. Lectin binding studies also suggested the presence of a terminal beta-galactose and GlcNAc in the glycan-inositol lipid core of LPG. Virtually all of the sialic acids appeared to be located in the saccharide portion of the molecule. Treatment of the LPG with phosphatidylinositol-specific phospholipase C liberated an alkylacylglycerol. Structural analysis of the alkylacylglycerol and its acidic methanolysis products by gas-liquid chromatography/mass spectrometry indicated that the glycerol substituents were primarily the C16 1-alkyl group and C16 2-acyl group. The ratio of inositol to 1-O-alkyl-2-O-acylglycerol was 1:1. Treatment of the glycoconjugate with nitrous acid released a major phospholipid product that migrated close to the phosphatidylinositol standard on thin layer chromatography. This result implied that phosphatidylinositol was glycosidically linked to the nonacetylated amino sugar. Furthermore, the LPG was found to contain phosphate and was labile to mild acid hydrolysis, strongly suggesting that the intact molecule is related to Leishmania LPG. The most striking and unique feature of T. cruzi LPG is the presence of large amounts of glucosamine and sialic acid as well as galactosamine. These results indicate that the glycoconjugate expressed on the T. cruzi cell surface is a new type of LPG-like molecule anchored on the cell surface via an alkylacylphosphatidylinositol.
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PMID:Expression of a novel cell surface lipophosphoglycan-like glycoconjugate in Trypanosoma cruzi epimastigotes. 807 17

Since glycosylphosphatidylinositol is the most common form of attachment of proteins to membranes in T. cruzi, and that this parasite depends on surface-mediated interactions for survival within the vector and mammalian host, it is probable that a drug which interfers with the metabolism of glycosylphosphatidylinositol (GPI) could be successfully employed in chemotherapy. Over the last few years several groups have been characterizing this mode of attachment in T. cruzi and more recently we have been concentrating our efforts on the identification of candidate precursors for protein anchors in metacyclic trypomastigotes. Previously detected GPI heterogeneity regarding solubilization of a major stage-specific antigen (1G7-Ag) by phospholipase C led us to investigate whether biosynthetic precursors with similar properties could also be identified. Two glycolipid species whose migration properties resemble glycolipids A and C of T. brucei were amenable to biosynthetic radiolabelling with palmitic acid, inositol, ethanolamine, glucosamine and mannose. Following purification, these species were submitted to classical GPI diagnostic treatments. In both cases digestion with GPI-specific phospholipase D (GPIPLD) produced phospatidic acid and treatment with either mild base or phospholipase A2 (PLA2) produced free fatty acid, indicating an acylation at least at position 2 of the glycerol. The glycolipid A-like species proved to be susceptible to solubilization by PIPLC of B. thuringiensis and by GPIPLC of T. brucei and the glycolipid C-like material proved to be fully resistant to both lipases.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Characterization of a phospholipase C-resistant inositol containing glycolipid from Trypanosoma cruzi. 808 Dec 35

Labelling with [3H]glucosamine was used to prepare a transforming growth factor-beta 1 (TGF-beta 1)-sensitive glycosylphosphatidylinositol (GPI) from monolayer cultures of rabbit articular chondrocytes (RAC), which may be involved in control of the cell cycle. The polar headgroup of this glycosylphosphatidylinositol was generated by both phosphatidylinositol-specific phospholipase C (PI-PLC) and pronase E digestion. The molecule emerged in only one peak on a Dowex AG1-X8 chromatogram, eluted at 0.1 N ammonium formate. In contrast, similar experiments performed on cellular extract from cultures previously labelled with [3H]glucosamine displayed four radioactive peaks eluting at 0.1, 0.2, 0.5 and 1 N ammonium formate, respectively. Evidence that the eluting position of these peaks was dependent on the number of phosphate residues present in each fraction was demonstrated by both [32P]phosphorus labelling and change in the position of alkaline phosphatase-induced shift in elution volume. We also demonstrated that the GPI-derived inositolphosphate glycan (IPG) could be hyperphosphorylated into the cell under the action of a kinase whose activity was enhanced by TGF-beta 1 itself. We have also shown that all of these IPG forms could mimic the TGF-beta-induced increase of DNA replication rate of RAC, with a higher activity for peaks III and IV than peaks I and II.
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PMID:Different phosphorylated forms of inositolphosphate glycan could be involved in the transforming growth factor-beta 1 (TGF-beta 1) signalling pathway. 808 80

Receptor-mediated activation of phospholipase C (PLC) which releases diacylglycerol and inositol trisphosphate has been implicated in the action of gonadotropin-releasing hormone (GnRH) on gonadotrophs. Previously we demonstrated that the synthetic diacylglycerol, phorbol 12-myristate 13-acetate (PMA) and PLC mimic the stimulatory effects of GnRH on both luteinizing hormone (LH) glycosylation and release. In this study we further investigated how PMA or PLC interact with GnRH to control LH release versus glycosylation. Cultured pituitary cells were incubated in the presence of radiolabeled precursors and GnRH (0, 1, or 100 nM), with or without PMA (10 nM) or PLC (0.24 U/ml) for 4 h. LH translation and glycosylation were monitored by measuring incorporation of [14C]alanine and [3H]glucosamine, respectively, into total (cell and medium) immunoprecipitable LH. Immunoreactive LH (IRLH) was measured by radioimmunoassay. Both PMA and PLC increased (p < 0.01) basal IRLH release, and IRLH release stimulated by 1 nM GnRH. Neither PMA nor PLC exerted an additive effect on IRLH release stimulated by 100 nM GnRH. The interactions between PMA or PLC and GnRH on IRLH release were significant (p < 0.01). Both PMA and PLC elevated (p < 0.01) total [3H]glucosamine-LH, but had no additive effect with 1 nM GnRH; PLC depressed (p < 0.05) the stimulatory effect of 100 nM GnRH, whereas PMA had no effect. The interactions between PMA or PLC and GnRH on LH glycosylation were significant (p < 0.01). PMA, PLC or GnRH alone did not affect total [14C]alanine-LH. In the presence of 1 or 100 nM GnRH, PLC, but not PMA, decreased (p < 0.05) total [14C]alanine-LH.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Differential actions of phospholipase C on gonadotropin-releasing-hormone-stimulated release and glycosylation of luteinizing hormone in rat anterior pituitary cells. 809 Feb 83

The lipid moiety of the lipophosphoglycan (LPG)-like glycoconjugates of Trichomonas vaginalis and Trichomonas foetus, parasites of the urogenital tract of human and cattle, respectively, has been isolated and characterized by a combination of enzymatic and chemical degradation, chromatography, and mass spectrometry. The carbohydrate composition of the glycan inositol lipid core is also reported. The glycan inositol core of trichomonad glycoconjugates is unique in having more than one GlcN and is significantly larger than any other glycan core reported so far. T. vaginalis glycoconjugate binds strongly to the lectin RCA-I, which suggest that the macromolecule possesses terminal beta 1,4-linked galactosyl residues. The binding of T. foetus glycoconjugate to the lectin UEA-I suggests the presence of terminal alpha 1,2-linked fucose. Acid hydrolysis of deaminated and reduced LPG products yields a [3H]anhydromannitol-containing product, indicating the presence of unacetylated glucosamine in the trichomonad LPGs. Reductive radiomethylation has been applied to label free amino groups in the hexosamine or other free amine-containing residues of the trichomonad glycoconjugates. Treatment of the LPGs with phosphatidylinositol-specific phospholipase C from Bacillus thuringiensis liberates a ceramide substituent. Treatment of LPGs with nitrous acid releases a phospholipid moiety containing myo-inositol and ceramide, implying that the LPGs are anchored in the membrane via an inositol-phosphate-ceramide. Structural characterization of the ceramide by gas-liquid chromatography (GC) and GC-mass spectrometry indicated the presence of the major long-chain base sphinganine (d 18: 0 dihydrosphingosine) and a C 16:0 N-acyl group. Lipophosphoglycans from both parasites contain ceramide as their only lipid moiety. These results suggest that T. vaginalis and T. foetus anchor their LPG-like glycoconjugates on the cell surface via inositol-phosphoceramide and also the glycan inositol core of the macromolecule appears to be unique in nature.
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PMID:Identification of the lipid moiety and further characterization of the novel lipophosphoglycan-like glycoconjugates of Trichomonas vaginalis and Trichomonas foetus. 813 38

The role of glycosyl-PtdIns (GPI)-anchored proteins in the cytodifferentiation of Tetrahymena vorax was examined. Labelling of cells with [3H]myristate or [3H]palmitate followed by electrophoresis showed an array of proteins carrying covalently bound lipids. Electrophoresis of protein from cells labelled with the GPI-anchor components [3H]Ins and [14C]ethanolamine revealed three polypeptides on fluorograms which have apparent molecular masses of approx. 28, 50 and 82 kDa. Labelled lipid associated with these polypeptides was susceptible to release by in vitro exposure to Bacillus thuringiensis PtdIns-specific phospholipase C (PI-PLC). Using labelled fatty acids, cells induced to differentiate showed altered GPI-anchored protein-labelling patterns in comparison with undifferentiated control cells, with a heavily labelled 32 kDa band appearing upon differentiation. Pre-incubation of cells in 10 mM D-mannosamine, an inhibitor of GPI incorporation into protein, resulted in a reduction of the incorporation of label into the three GPI-anchored proteins, nearly complete inhibition of differentiation and a reduction in the rate of digestive vacuole formation. A 50% inhibition of differentiation was obtained using 500 microM mannosamine. The inhibitory impact of D-mannosamine on differentiation could be competitively and completely reversed by the inclusion of D-mannose, but not D-glucose. Neither glucosamine nor tunicamycin inhibited differentiation. Incubation of cells in PI-PLC (5 units/ml) plus the differentiation inducer resulted in an acceleration of differentiation and generally higher percentages of differentiated cells versus controls.
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PMID:Cytodifferentiation in Tetrahymena vorax is linked to glycosyl-phosphatidylinositol-anchored protein assembly. 814 85

In this study we describe the biochemical features of the Toxoplasma gondii tachyzoite surface glycoprotein, gp23, demonstrating that it is attached to the parasite membrane by a glycosyl-phosphatidyl inositol anchor. Gp23 was metabolically labeled with tritiated palmitate, myristate, ethanolamine, inositol, glucosamine, mannose and galactose, as expected for a GPI-anchor structure. Gp23 was released from the surface of living parasites after treatment with phosphatidylinositol-specific phospholipase C(PI-PLC) and the resulting water-soluble protein was immunoprecipitated with a monoclonal antibody specific for gp23. The GPI-core glycan was generated after aqueous-HF dephosphorylation followed by nitrous acid deamination and its carbohydrate structure was analyzed using selective exo- and endoglycosidase treatments. Finally, the phosphatidylinositol moiety of gp23 was characterized using PI-PLC and phospholipase A2 (PLA2) digestions. Our cumulative data suggest that gp23 of T gondii tachyzoites contains a modified GPI-backbone similar to the mammalian Thy-1 anchor, consisting of a conserved core structure (ethanolamine-PO4-6-Man alpha1-2-Man alpha1-6-Man alpha1-4-GlcN alpha1-6-PI) bearing beta-linked N-acetylgalactosamine residue(s).
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PMID:Structural analysis of glycosyl-phosphatidylinositol membrane anchor of the Toxoplasma gondii tachyzoite surface glycoprotein gp23. 824 58

Plasmodium falciparum is the causative agent of malaria tropica in man. Biochemical studies were focused on the asexual, intraerythrocytic stages of P. falciparum, because of their role in the clinical phase of the disease and the possibility of propagation in a cell culture system. In this report, we describe the in-culture labeling of malarial glycolipids and the analysis of their hydrophilic moieties. They were identified as glycosylphosphatidylinositols (GPIs) by: 1) labeling with [3H]mannose, [3H]glucosamine, and [3H]ethanolamine and 2) sensitivity toward glycosylphosphatidylinositol-specific phospholipase D, phospholipase A2, and nitrous acid. Malarial GPIs are shown to be unaffected by treatment with phosphatidylinositol-specific phospholipase C, regardless of prior treatment with mild base commonly used for inositol deacylation. Two candidates for putative GPI-anchor precursors to malarial membrane proteins with the structures ethanolamine-phosphate-6(Man alpha 1-2)Man alpha 1-2Man alpha 1-6Man alpha 1-4 GlcN-PI (Pfg1 alpha) and ethanolamine-phosphate-6Man alpha 1-2Man alpha 1-6Man-alpha 1-4-GlcN-PI (Pfg1 beta) were identified.
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PMID:Glycosylphosphatidylinositols synthesized by asexual erythrocytic stages of the malarial parasite, Plasmodium falciparum. Candidates for plasmodial glycosylphosphatidylinositol membrane anchor precursors and pathogenicity factors. 830 May 89

In our study, 5'-nucleotidase was released from bovine liver by the treatment with Bacillus thuringiensis phosphatidylinositol-specific phospholipase C and purified to a homogeneous state by concanavalin A-Sepharose and (diethylaminoethyl)-Toyopearl column chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Purified 5'-nucleotidase were then cleaved by cyanogen bromide (CNBr), and then inositol phosphoglycan-containing C-terminal peptides (IPG peptides) were separated by C18 reverse-phase liquid chromatography and analyzed by peptide sequencer, amino acid analyzer, gas chromatography (GC), and GC-mass spectrometry (MS). Ser523 of the amino acid sequence deduced from 5'-nucleotidase cDNA [Suzuki et al. (1993) J. Biochem. (Tokyo) 113, 607-613] is revealed to be the C-terminal amino acid to which a glycosylphosphatidylinositol is anchored. Separated peaks of CNBr-cleaved IPG peptides were then analyzed by electron spray ionization (ESI)-MS. Eight different molecular weight (MW) species of CNBr-cleaved IPG peptides were detected. Three fractions of CNBr-cleaved IPG peptides were separately treated by trypsin, and trypsinized IPG peptides were purified by C18 reverse-phase liquid chromatography. Finally, five different MW species of trypsinized IPG peptides (1629.4, 1752.7, 1791.8, 1832.8, and 1994.5) were detected by ESI-MS. Together with sequential exoglycosidase treatment and quantitative analysis of sugar moieties by GC and GC-MS, microheterogeneity in the structures of these five glycosylphosphatidylinositol (GPI) anchor species was determined. The common core structure was ethanolamine phosphate-mannose-mannose-mannose(-ethanolamine phosphate)-glucosamine-myoinositol phosphate. Variations observed in additional mannose, N-acetylhexosamine, and ethanolamine phosphate moieties form this heterogeneity.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Microheterogeneity in glycosylphosphatidylinositol anchor structures of bovine liver 5'-nucleotidase. 830 28

An inositol phosphoglycan that is the polar head group of a glycosyl phosphatidylinositol has been considered as a putative mediator of insulin action. To gain insight into the functions of this hormone during development, the relationships between insulin, insulin receptors, glycosyl phosphatidylinositol, and inositol phosphoglycan were studied. Glycosyl phosphatidylinositol was isolated and characterized in fetal liver as early as day 15 of intrauterine life. In isolated hepatocytes from fetal and adult rats labeled with [3H]glucosamine, [3H]galactose, or [3H]myo-inositol, these molecules were incorporated into glycosyl phosphatidylinositol. In hepatocytes labeled with [3H]glucosamine and then allowed to react with [1-14C]IAI, the [3H]glycosyl phosphatidylinositol was purified as the 14C-labeled amidinated lipid. Glycosyl phosphatidylinositol molecules from fetal and adult cells were sensitive to hydrolysis by a phosphatidylinositol-specific phospholipase C from B. cereus. The product of this hydrolysis inhibits the activity of a cAMP-dependent protein kinase, whereas this effect was abolished by nitrous acid deamination. In isolated hepatocytes from adult animals, an inverse correlation between extracellular insulin and the number of insulin receptors and the cellular content of glycosyl phosphatidylinositol was observed. However, in fetal hepatocytes insulin failed to reduce the glycosyl-phosphatidylinositol content when labeled either with [1-14C]isethionyl acetimidate or [3H]glucosamine, whereas insulin-like growth factor I produced a significant hydrolysis of glycosyl phosphatidylinositol. Fetal and adult hepatocytes were incubated with insulin or inositol phosphoglycan after which glycogen phosphorylase activities were determined. Inositol phosphoglycan mimicked the action of insulin on both forms of the enzyme from adult hepatocytes, whereas in fetal cells insulin did not change, and purified inositol phosphoglycan reduced the activities of glycogen phosphorylase. These findings suggest a dissociation between insulin receptor occupancy and the expected hormonal effects in fetal hepatocytes. This could be related to alterations at a postreceptor level.
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PMID:Insulin does not induce the hydrolysis of a glycosyl phosphatidylinositol in rat fetal hepatocytes. 834 37


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