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:3.5.1.4 (deaminase)
5,113 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The glycoprotein nature of the binding subunit of the dopamine D2 receptor in rat striatum has been examined by photoaffinity labeling receptor preparations with N-(p-azido-m-[125I]iodophenethyl)spiperone followed by treatment of crude membrane receptor or receptor fractions isolated from sodium dodecyl sulfate (SDS) polyacrylamide gels with endo- and exoglycosidases. The major photoaffinity labeled protein migrates as a heterogeneous species on 10% SDS polyacrylamide gels and ranges from 130,000 to 75,000 relative molecular mass (Mr). This heterogeneity can be explained by glycosylation of the receptor by complex-type N-linked oligosaccharides. Three fractions of labeled receptor were isolated from SDS polyacrylamide gels over a range of 130,000 to 75,000 Mr; after digestion with peptide-N4-[N-acetyl-beta-glucosaminyl] asparagine amidase, all fractions yielded a single peptide approximately 40,000 Mr. Treatment of photoaffinity labeled membranes with alpha-mannosidase was without effect. The dopamine D2 receptor appears to contain substantial amounts of sialic acid as treatment of photoaffinity labeled membranes with neuraminidase increased the receptor mobility on SDS polyacrylamide gels to a species of 50,000-54,000 Mr. Treatment of the receptor with neuraminidase followed by endo-alpha-N-acetylgalactosaminidase did not change the electrophoretic migration pattern from that seen after neuraminidase treatment alone, suggesting that the binding peptide contains no serine- or threonine-linked oligosaccharides. A smaller binding peptide of approximately 31,000 Mr is also apparent in crude photoaffinity labeled membranes. This material also contains N-linked oligosaccharide. Complete removal of N-linked oligosaccharide from the dopamine D2 receptor did not change the rank order potency of agonist and antagonist compounds to compete for [3H]spiperone binding to crude membrane fractions. The dopamine D2 receptor represents a highly glycosylated neural receptor.
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PMID:N-linked oligosaccharides are responsible for rat striatal dopamine D2 receptor heterogeneity. 252 26

Transfer of truncated oligosaccharides to protein in vivo and the structure of Man2GlcNAc2 synthesized by intact yeast (Saccharomyces cerevisiae) were investigated in the alg2 mutant. At the nonpermissive temperature the alg2 mutant accumulates lipid-linked oligosaccharides that migrate on Bio-Gel P4 in the range expected for Man2GlcNAc2 and Man1GlcNAc2 (T.C. Huffaker and P.W. Robbins (1983) Proc. Natl. Acad. Sci. USA 80, 7466-7470). We characterized the oligosaccharides, derived from protein and lipid, by comigration with standards on HPLC and by Smith degradation followed by HPLC. Man2GlcNAc2 and Man1GlcNAc2 are found on protein in alg2, since their release from a protein-containing precipitate of alg2 cells is N-glycanase (peptide-N4[N-acetyl-beta-glucosaminyl]asparagine amidase) dependent. Transfer also occurred in alg2/pAC3 cells, which carry ALG2 on a multicopy plasmid that confers partial correction of the oligosaccharide phenotype. The alg2/pAC3 cells are viable at 36 degrees C. Two isomers of Man2GlcNAc2, Man1----3ManGlcNAc2 and Man1----6ManGlcNAc2, were present on lipid and protein. The transfer of Man2GlcNAc2 and Man1GlcNAc2 to protein by intact cells supports topological models that postulate access by early intermediates to the lumen of the endoplasmic reticulum.
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PMID:Synthesis of lipid-linked oligosaccharides in Saccharomyces cerevisiae: Man2GlcNAc2 and Man1GlcNAc2 are transferred from dolichol to protein in vivo. 266 Jul 43

To examine the function of the carbohydrate chains of cobra venom factor (CVF), the molecule was enzymatically deglycosylated under non-denaturing conditions with N-glycanase (peptide-N4-(N-acetyl-beta-glucosaminyl) asparagine amidase). The deglycosylation of CVF chains seems to proceed independently of each other, leading to partially deglycosylated intermediates. Complete deglycosylation of CVF was found to abolish the activity of CVF. The deglycosylated molecule is unable to activate the alternative pathway of complement. Deglycosylated CVF no longer consumes the serum complement activity, it does not induce C3 activation in serum, nor does it induce complement-mediated hemolysis. These results indicate that the carbohydrate moieties of CVF are essential for its role in complement activation.
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PMID:The oligosaccharide chains of cobra venom factor are required for complement activation. 277 Jul 49

The binding subunit of the alpha 1-adrenergic receptor has been identified as an Mr = 80,000 peptide in several tissues. Adsorption of the alpha 1-adrenergic receptor to a wheat germ agglutinin lectin-agarose resin suggests that the receptor protein is glycosylated. In this study, we investigated the nature of the carbohydrate chains linked to the alpha 1-adrenergic receptor peptide. The alpha 1-adrenergic receptor from DDT2 MF-2 smooth muscle cell and rat brain membranes was photolabeled with 125I-azido-prazosin [( 125I]CP65,526) and then treated with exoglycohydrolases prior to SDS-PAGE and autoradiography. Removal of terminal sialic acid residues by neuraminidase decreased the receptor Mr by 6,000; however, alpha-mannosidase was without effect, indicating complex type glycosylation of the receptor-protein. Similar results were observed for the rat hepatic membrane alpha 1-adrenergic receptor. Removal of N-linked carbohydrates at asparagine residues by peptide-N4[N-acetyl-beta-glucosaminyl]asparagine amidase (from Flavobacterium meningosepticum) resulted in a specifically labeled peptide at Mr = 50,000-55,000 in DDT1 MF-2 membrane and solubilized receptor preparations. Treatment of DDT1 MF-2 cells with swainsonine or (+)-1-deoxymannojirimycin, inhibitors of complex type carbohydrate chain biosynthesis, caused a reduction in the apparent molecular weight of the receptor (Mr = 60,000) but did not alter the number of alpha 1-adrenergic receptors per cell or their affinity for the radioligand [3H]prazosin. These findings indicate that the alpha 1-adrenergic receptor is heavily glycosylated, the major oligosaccharide moiety being of the complex type, N-linked to asparagine residues. The peptide backbone of the receptor has an Mr less than or equal to 55,000, consistent with the predicted molecular mass of other membrane neurotransmitter receptors based on sequence analysis of isolated cDNA clones.
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PMID:Glycosylation of the mammalian alpha 1-adrenergic receptor by complex type N-linked oligosaccharides. 282 78

Human hepatoma (Hep G2) cells secrete nanogram quantities of carboxypeptidase enzymes which are capable of hydrolyzing COOH-terminal lysine and arginine residues. A carboxypeptidase with a neutral pH optimum (greater than pH 7.0) was partially purified from the conditioned medium and compared with pure plasma carboxypeptidase N. The two enzymes behaved in a similar manner on gel filtration (apparent Mr = 280,000), DE52 ion exchange chromatography, and concanavalin A-affinity chromatography and were indistinguishable enzymatically and immunologically. Immunoblots of the Hep G2 and plasma carboxypeptidase N before and following deglycosylation with peptide-N4-[N-acetyl-beta-glucosaminyl]asparagine amidase F revealed a similar, if not identical, multimeric structure. A second carboxypeptidase with a lower molecular weight and a pH optimum of 5.0 was also detected in the Hep G2 medium.
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PMID:Characterization of the carboxypeptidase N secreted by Hep G2 cells. 284 69

The ligand-binding subunit of the porcine striatal dopamine D2 receptor was identified by photoaffinity labeling with [125I]N-azidophenethylspiperone ([125I]NAPS). Upon photolysis, [125I]NAPS covalently incorporated into a broad band of apparent Mr congruent 140,000 with an appropriate pharmacological profile for D2 receptors as assessed by autoradiography after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Smaller subunits of apparent Mr congruent 94,000 and 34,000 were specifically labeled by [125I]NAPS with an appropriate D2 receptor profile and were similar to the major ligand-binding subunits of photoaffinity-labeled canine striatal D2 receptors. Photoaffinity labeling in the absence or presence of multiple protease inhibitors did not alter the migration pattern of the Mr congruent to 140,000/94,000 subunits upon denaturing electrophoresis in either the absence or presence of thiol-reducing/alkylating reagents. In order to investigate the possible basis for the existence of these high molecular weight forms of the D2 receptor, we assessed the carbohydrate nature of photolabeled D2 ligand-binding subunits by the use of lectin affinity chromatography and specific exo- and endoglycosidase treatments. Both photoaffinity-labeled D2 receptor proteins from porcine striatum (Mr congruent to 140,000 and 94,000) were glycoproteins as indexed by their absorption and specific elution from wheat germ agglutinin lectin resins. The exoglycosidase neuraminidase altered the electrophoretic mobility of both the Mr congruent to 140,000 and 94,000 labeled subunits to a single band of apparent Mr congruent to 51,000. Prior removal of sialic acid residues did not alter the reversible binding characteristics of [3H]spiperone to D2 receptors. Complete removal of receptor-associated N-linked carbohydrate by the endoglycosidase glycopeptidase F (peptide-N4[N-acetyl-beta-glucosaminyl]asparagine amidase) produced a further increase in the mobility of the Mr congruent to 51,000 subunit to apparent Mr congruent to 44,000. The porcine Mr congruent to 34,000 photolabeled peptide is an N-linked glycoprotein as assessed by lectin affinity chromatography and susceptibility to digestion by glycopeptidase F to a peptide of apparent Mr congruent to 23,000.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Dopamine D2 receptor binding subunits of Mr congruent to 140,000 and 94,000 in brain: deglycosylation yields a common unit of Mr congruent to 44,000. 297 May 86

Enzymatic deacylation of the lipopolysaccharide isolated from a Salmonella Rd mutant by a cell-free preparation from Acanthamoeba castellanii has been studied. The degradation was found to be dependent on the presence of a surface-active component (Triton X-100) in the reaction mixture. The lipid A part of the lipopolysaccharide was the primary target of the enzymes, which cleaved with high efficiency the ester-bound long-chain nonhydroxylated and 3-hydroxylated acyl residues, i.e. lauric, myristic, palmitic and 3-hydroxymyristic acid. The cell-free preparation also exhibited amidase activity cleaving about 50% of the amide-bound 3-hydroxymyristic acid residues. In addition the extract proved to possess phosphatase activity liberating ester-bound and glycosidically bound phosphate groups of lipid A. On the other hand, the glucosaminyl-beta 1,6-glucosamine disaccharide was not degraded and remained bound to the oligosaccharide part (heptose/3-deoxyoctulosonic acid) of the lipopolysaccharide.
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PMID:In vitro deacylation of lipopolysaccharide of Salmonella minnesota by Acanthamoeba castellanii enzymes. 300 30

Fibrinogen, the major structural precursor of blood clots, was deglycosylated by peptide-N-(N-acetyl-beta-glucosaminyl)asparagine amidase without denaturation of the polypeptide chains. Deglycosylated fibrinogen behaved normally in clinical coagulation assays, although it is less soluble than normal fibrinogen. However, the turbidity of clots formed from deglycosylated fibrinogen always rose faster and higher than that of clots from normal fibrinogen. Scanning and transmission electron microscopy demonstrated that fibrin made from clots of deglycosylated fibrinogen consisted of thicker, less-branched fiber bundles in a more porous network. Moreover, the degree of lateral aggregation was directly related to clot turbidity and inversely related to branching. Deglycosylation promoted turbidity development, lateral aggregation, and porosity of clots under all conditions tested. All other steps in the coagulation pathways appeared to be unaffected by the absence of carbohydrate. These results suggest that carbohydrate constitutively affects the behavior of deglycosylated fibrinogens by 1) contributing a repulsive force that promotes fibrinogen solubility and limits fibrin assembly and 2) sensitizing fibrin to conditions that influence assembly and clot structure.
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PMID:Deglycosylation of fibrinogen accelerates polymerization and increases lateral aggregation of fibrin fibers. 317 May 75

Aminooligopeptidase is an intrinsic glycoprotein of the brush border membrane important for hydrolysis of the oligopeptide products of intraluminal protein digestion. To study its synthesis and intracellular processing, we performed pulse-chase experiments using [35S]methionine to label proteins of cultured human intestinal explants obtained by endoscopic biopsy. Aminooligopeptidase was isolated by immune precipitation with a monoclonal antibody and its molecular size was assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography. A precursor of relative molecular weight (Mr) 127,000 appeared within 10 min of chase and appeared to begin conversion to an Mr 150,000 form (the size of brush border membrane aminooligopeptidase) within 60 min. To determine if the change in molecular size was the consequence of alterations in glycosylation, we studied the susceptibility of the two forms to endo-beta-N-acetylglucosaminidase H, which cleaves immature high-mannose N-linked carbohydrate chains, and to peptide: N4-(N-acetyl-beta-glucosaminyl)asparagine amidase, which cleaves both the high-mannose and complex N-linked carbohydrate chains. Only the early Mr 127,000 aminooligopeptidase was sensitive to endo-beta-N-acetylglucosaminidase H, suggesting that the larger form results from trimming of high-mannose cores and adding terminal sugars in the Golgi complex. Both forms were sensitive to peptide: N4-(N-acetyl-beta-glucosaminyl)asparagine amidase, generating an Mr 114,000 species. The kinetics of the synthesis and processing of aminooligopeptidase and sucrase-isomaltase were compared by immunoprecipitation of both proteins from the same tissue after separating the microvillous membrane from the remainder of the cellular membranes. Labeled aminooligopeptidase was present intracellularly in its mature form within 60 min and was detected exclusively in the brush border membrane by 90 min. Most of the labeled sucrase-isomaltase pool had not yet undergone complex glycosylation during the same period. These data demonstrate that although human intestinal aminooligopeptidase undergoes N-linked glycosylation like sucrase-isomaltase, the synthesis of aminooligopeptidase differs from that of sucrase-isomaltase in respect to the absence of a high-molecular-weight precursor and more rapid pre-Golgi processing.
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PMID:Synthesis and intracellular processing of aminooligopeptidase by human intestine. 336 Feb 63

An enzymatic procedure for releasing asparagine-linked oligosaccharides from glycoproteins by treatment with N-glycanase (peptide-N4-(N-acetyl-beta-glucosaminyl) asparagine amidase) has been investigated. Ribonuclease B, transferrin, fetuin, and alpha 1-acid glycoprotein were treated with N-glycanase and the released oligosaccharides were radiolabeled with NaB3H4. Lectin staining of the N-glycanase-treated proteins indicated that the deglycosylation reactions had proceeded to completion. The labeled carbohydrate chains were analyzed by HPLC on Micro-Pak AX-5 and AX-10 columns. The proportion of high-mannose and bi-, tri-, and tetraantennary complex chains obtained from each glycoprotein was in agreement with literature values. These results demonstrate that N-glycanase provides a simple method to release all common classes of asparagine-linked oligosaccharides from a glycoprotein in a form that can be radiolabeled directly for structural analysis.
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PMID:Use of N-glycanase to release asparagine-linked oligosaccharides for structural analysis. 360 11


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