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

---

Query: EC:3.1.4.3 (phospholipase C)
18,461 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Decay-accelerating factor (DAF) is anchored in cell membranes by a glycosyl-plasmanylinositol (GPI) moiety that is transferred to it en bloc in the rough endoplasmic reticulum. To analyze the biochemical reactions involved in preassembly of this structure, a human hematopoietic cell-free system was employed. Incubation of cell extracts with UDP-[3H]GlcNAc and butanol partitioning of reaction mixtures yielded two products similar in TLC mobility to intermediates described in Trypanosoma brucei. Both species were sensitive to Bacillus thuringiensis phosphatidylinositol-specific phospholipase C, indicative of association of [3H]GlcNAc label with a plasmanylinositol-containing acceptor. In contrast to trypanosome intermediates, which contain phosphatidylinositol (1,2-diacylglycerophosphoinositol), however, alkali treatment and phospholipase A2 digestion generated butanol-phase products characteristic of glycosylated plasmanylinositol (1-alkyl-2-acylglycerophosphoinositol). Kinetic and pulse-chase experiments indicated that the slower-migrating species was a product of the faster and that it, but not the faster, was sensitive to both GPI-specific phospholipase D and nitrous acid deamination, consistent with conversion of GlcNAc- to GlcN-plasmanylinositol. Accordingly, acetic anhydride acetylation retransformed the slower species back to the faster. Further incubation with cell extracts converted the slower species into more polar products. Lysates of normal and of affected blood leukocytes from two paroxysmal nocturnal hemoglobinuria (PNH) patients supported assembly of the two intermediates within 1 min. Thus, the initial enzymes mediating human GPI-anchor assembly are GlcNAc-plasmanylinositol transferase and GlcNAc-plasmanylinositol deacetylase, their substrates contain plasmanylinositols, and the products of their activities are normal in affected PNH cells.
...
PMID:Assembly and deacetylation of N-acetylglucosaminyl-plasmanylinositol in normal and affected paroxysmal nocturnal hemoglobinuria cells. 170 86

Four monoclonal antibodies (MAbs) specific for Trypanosoma cruzi were obtained. Flow cytometry analysis showed that these four MAbs stained the membranes of the three main morphological forms of T. cruzi: amastigotes, trypomastigotes, and epimastigotes. The four MAbs seemed to recognize the same 50- to 55-kDa antigen that was revealed by immunoblotting. Competition experiments revealed that they defined at least two different epitopes on the molecule. The antigen was detected on the external surface of the membrane by immunoelectron microscopy. Several experiments indicated that the 50- to 55-kDa antigen recognized by these four MAbs was a glycosyl-phosphatidylinositol-anchored membrane protein. (i) The antigen could be removed from the cell surface by treatment with proteases, NaOH, HNO2, and phosphatidylinositol-specific phospholipase C (PI-PLC). (ii) The phase distribution of the antigen in Triton X-114 solutions changed drastically upon treatment with PI-PLC. The antigen was found mainly in the detergent phase in nontreated samples and in the aqueous phase in PI-PLC-digested samples. (iii) A cross-reacting determinant that was found in other glycosyl-phosphatidylinositol-anchored membrane proteins appeared after PI-PLC treatment.
...
PMID:Characterization of a glycosyl-phosphatidylinositol-anchored membrane protein from Trypanosoma cruzi. 182 89

The glycosyl phosphatidylinositol (GPI) anchors that attach certain proteins to membranes are preassembled by sequential addition of glycan components to phosphatidylinositol (PI) before being transferred to nascent polypeptide. A cell-free system consisting of trypanosome membranes has been reported to catalyze GPI biosynthesis (Masterson, W. J., Doering, T. L., Hart, G. W., and Englund, P. T. (1989) Cell 56, 793-800; Menon, A. K., Schwarz, R. T., Mayor, S., and Cross, G. A. M. (1990) J. Biol. Chem. 265, 9033-9042). We now describe conditions for studying the initial steps of GPI biosynthesis in extracts of murine lymphoma cells. Two chloroform-soluble products, tentatively identified as [6-3H]GlcNAc-PI and [6-3H]GlcN-PI were generated during incubations of EL4 cell lysates with UDP-[6-3H]GlcNAc. The involvement of PI in the reaction was established by the sensitivity of the products to hydrolysis by PI-specific phospholipase C and the finding that the addition of exogenous PI to the incubation stimulated the reaction. The minor, more polar product was sensitive to nitrous acid cleavage and was converted to the major product, as judged by TLC, after treatment with acetic anhydride. The glycolipids generated in lymphoma extracts appeared to be the same as the products produced in parallel incubations with trypanosome membranes. Analysis of available lymphoma mutants deficient in Thy-1 surface expression revealed that extracts of the class A, C, and H mutants are completely defective in synthesizing GlcNAc-PI and GlcN-PI.
...
PMID:Defective glycosyl phosphatidylinositol biosynthesis in extracts of three Thy-1 negative lymphoma cell mutants. 182 68

The insoluble residue from Tetrahymena mimbres cells that had been preincubated in vivo for 2 h with [3H]myristic acid and then exhaustively delipidated with organic solvents retained radioactivity, principally in material which migrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent molecular mass of 10-14 kDa. This material was extractable from the delipidated cell residue with organic solvents known to solubilize phosphatidylinositol glycans (PI glycans). The same material could also be labeled with [3H]inositol, [14C]glucosamine, and [3H] ethanolamine. When the delipidated residue of cells labeled for 2 h with [3H]myristate was treated with phosphatidylinositol-specific phospholipase C or nitrous acid, much of the associated radioactivity was released. A similar release was obtained using the putative PI glycan fraction extracted from the cell residue. After further purification by thin layer chromatography, this latter material was hydrolyzed with HCl and shown to contain fatty acids, alkylglyceryl ethers, phosphate, inositol, glucosamine, mannose, and ethanolamine. The findings indicate that T. mimbres contains PI glycans resembling in structure those recently characterized in trypanosomes and mammalian cells. As the time of incubation with the radiotracers enumerated above was increased to 6-24 h, increasing amounts of radioactivity appeared in the 22-27-kDa region of sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels. This higher molecular weight material is shown in the companion paper (Pak, Y., Ryals, P.E., and Thompson, G.A., Jr. (1991) J. Biol. Chem. 266, 15054-15059) to be released by in vivo phosphatidylinositol-specific phospholipase C treatment. Thus T. mimbres contains a pool of free PI glycans and at least one phosphatidylinositol-anchored protein.
...
PMID:Phosphatidylinositol-linked glycans and phosphatidylinositol-anchored proteins of Tetrahymena mimbres. 186 41

Incubation of microsomal preparations from Leishmania donovani parasites with UDP-[3H]galactose or GDP-[14C]mannose resulted in incorporation of radiolabel into an endogenous product that exhibited the chemical and chromatographic characteristics of the parasite's major surface glycoconjugate, lipophosphoglycan. The [3H]galactose- or [14C]mannose-labeled product was (i) cleaved by phosphatidylinositol-specific phospholipase C; (ii) deaminated by nitrous acid; and (iii) degraded into radioactive, low molecular weight fragments upon hydrolysis with mild acid. Analysis of the products of mild acid hydrolysis revealed the presence of phosphorylated Gal-beta-Man as the major fragment with lesser amounts of mono-, tri-, and tetrasaccharides. The incorporation of the two isotopic precursors was neither stimulated by the addition of dolichylphosphate nor inhibited by amphomycin, indicating that dolichol-saccharide intermediates are not involved in assembly of the repeating units of lipophosphoglycan. Development of this cell-free glycosylating system will facilitate further studies on the pathway and enzymes involved in lipophosphoglycan biosynthesis.
...
PMID:Cell-free biosynthesis of lipophosphoglycan from Leishmania donovani. Characterization of microsomal galactosyltransferase and mannosyltransferase activities. 190 57

Glycosylated phosphatidylinositol (gly-Pl) molecules have been implicated as precursors for insulin-sensitive second messengers (1-4) and lipid-anchored membrane proteins (5-9). The relationship between the diverse functions of these lipids and their predicted structural heterogeneity within gly-Pl subtypes was examined in human T lymphocytes. Four subtypes of gly-Pl molecules were identified in T lymphocytes after separation over high-performance thin-layer chromatography by sensitivity to Pl-specific phospholipase C and nitrous acid. Antibody probes of the glycan domain of gly-Pl were developed and used to assess the partial sensitivity of gly-Pl to insulin action. This analysis showed that the effects of insulin are linked to differential utilization of only two of the four gly-Pl subtypes in T lymphocytes. Polar fragments of this reaction were identified in extracellular supernatants from insulin-treated cells. The biological significance of insulin-dependent gly-Pl hydrolysis was demonstrated by insulin and inositol phosphoglycan regulation of glucose metabolism in intact lymphocytes. These results support the hypothesis that multifunctional roles of gly-Pl are served by discrete gly-Pl populations and that metabolites of gly-Pl subsets participate as signaling elements in insulin action.
...
PMID:Differential regulation of glycosylated phosphatidylinositol subtypes by insulin. 193 92

Renal dipeptidase (EC 3.4.13.11) has been purified from human kidney cortex by affinity chromatography on cilastatin-Sepharose following solubilization with either n-octyl-beta-D-glucopyranoside or bacterial phosphatidylinositol-specific phospholipase C (PI-PLC). Phase separation in Triton X-114 revealed that the detergent-solubilized form was amphipathic and retained the glycosyl-phosphatidylinositol membrane anchor whereas the phospholipase solubilized form was hydrophilic. Both forms of the enzyme existed as a disulphide-linked dimer of two identical subunits of Mr 59,000 each. The glycosyl-phosphatidylinositol anchor of purified human renal dipeptidase was hydrolysed by a range of bacterial PI-PLCs and by a plasma phospholipase D. Mild acid treatment and nitrous acid deamination of the hydrophilic form revealed that the cross-reacting determinant, characteristic of the glycosyl-phosphatidylinositol anchor, was due exclusively to the inositol 1,2-cyclic phosphate ring epitope. The N-terminal amino acid sequences of the amphipathic and hydrophilic forms were identical, locating the membrane anchor at the C-terminus. The N-terminal sequence of human renal dipeptidase showed a high degree of similarity with that of the pig enzyme, and enzymic deglycosylation revealed that the difference in size of renal dipeptidase between these two species is due almost entirely to differences in the extent of N-linked glycosylation.
...
PMID:Characterization of the glycosyl-phosphatidylinositol-anchored human renal dipeptidase reveals that it is more extensively glycosylated than the pig enzyme. 213 35

Metabolic radiolabeling of adult worms of Schistosoma mansoni with [3H]myristic acid has revealed that the fatty acid is incorporated into more than 15 proteins. We have shown that two of these proteins, a 200-kDa glycoprotein known to be exposed on the surface of the adult worm following praziquantel treatment and a 22-kDa glycoprotein that shows an enhanced immune reactivity with sera of vaccinated mice, are anchored to the adult worm membrane via a glycosylphosphatidylinositol (GPI) linkage. Both antigens partitioned preferentially into the detergent phase of Triton X-114 and were susceptible, following immunoaffinity purification, to hydrolysis by phosphatidylinositol-specific phospholipase C (PIPLC) from Bacillus thuringiensis and phospholipase C from Bacillus cereus. Diacylglycerol (DAG) was released following hydrolysis by bacterial PIPLC; however, Trypanosoma brucei GPIPLC failed to release the diacylglycerol from either protein. Treatment with nitrous acid generated phosphatidylinositol (PI) from both proteins, and phospholipase D from rat serum cleaved phosphatidic acid from the 200-kDa protein. Although the functional significance of these GPI-anchored proteins is unknown, their release from the surface of the schistosome may contribute to immune evasion.
...
PMID:Identification and characterization of glycosylphosphatidylinositol-linked Schistosoma mansoni adult worm immunogens. 213 72

Aminopeptidase P (EC 3.4.11.9) was solubilized from pig kidney membranes with bacterial phosphatidylinositol-specific phospholipase C (PI-PLC) and then purified by a combination of anion-exchange and hydrophobic-interaction chromatographies. Contaminating peptidase activities were removed by selective affinity chromatography. The purified enzyme was apparently homogeneous on SDS/PAGE with an Mr of 91,000. Enzymic deglycosylation revealed that aminopeptidase P is a glycoprotein, with up to 25% by weight of the protein being due to the presence of N-linked sugars. The phospholipase-solubilized aminopeptidase P was recognized by an antiserum to the cross-reacting determinant (CRD) characteristic of the glycosyl-phosphatidylinositol anchor. This recognition was abolished by mild acid treatment or deamination with HNO2, indicating that the CRD was due exclusively to the inositol 1,2-cyclic phosphate ring epitope generated by the action of PI-PLC. The activity of aminopeptidase P was inhibited by chelating agents and was stimulated by Mn2+ or Co2+ ions, confirming the metallo-enzyme nature of this peptidase. Selective inhibitors of other aminopeptidases (actinonin, amastatin, bestatin and puromycin) had little or no inhibitory effect.
...
PMID:Purification and characterization of pig kidney aminopeptidase P. A glycosyl-phosphatidylinositol-anchored ectoenzyme. 213 78

The lipopeptidophosphoglycan (LPPG) from Trypanosoma cruzi, a major constituent of the plasma membrane of epimastigote forms, has been now extracted with butanol/water from delipidated cells and purified by hydrophobic chromatography. We have found that the LPPG undergoes two reactions, characteristic of the glycosylphosphatidylinositol anchors: (a) cleavage of the ceramide by phosphatidylinositol-specific phospholipase C (PtdIns-specific phospholipase C) from Bacillus thuringiensis, (b) nitrous acid deamination of the non-N-acylated glucosamine. Palmitoylsphinganine, palmitoylsphingosine, lignoceroylsphinganine and, as minor components, the stearoylceramides were identified by gas liquid chromatography/mass spectrometry. The presence of cross reacting determinant (CRD) epitopes in the glycophosphoinositol released by PtdIns-specific phospholipase C was investigated by direct and inhibition ELISA. A sample of glycophosphoinositol containing 5 micrograms carbohydrate caused 60% inhibition of the binding of anti-CRD antibodies raised against the soluble form of variant surface glycoprotein.
...
PMID:Structural features of the lipopeptidophosphoglycan from Trypanosoma cruzi common with the glycophosphatidylinositol anchors. 214 55


<< Previous 1 2 3 4 5 6 7 Next >>

---