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 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

A perfused rat liver was used to study the effects of 5-diazo-4-oxo-L-norvaline on lysosomal glycoprotein catabolism. Addition of this compound (1.0 mM) to the perfusate reduced activity of beta-aspartyl-N-acetylglucosylamine amidohydrolase by 99% in 1 h. Treated livers were unable to completely degrade endocytosed N-acetyl[14C]glucosamine-labeled asialo-alpha 1-acid glycoprotein as evidenced by a 50% reduction in radiolabeled serum glycoprotein secretion compared to controls. This decreased degradation was matched by a lysosomal accumulation of glycopeptides with the structure: GlcNAc beta(1-4)GlcNAc-Asn. The result suggested the presence of an unrecognized glycosidase in rat liver lysosomes, since this remnant was extended by one more GlcNAc residue than would have been expected after specific inactivation of the amidohydrolase. Such a novel enzyme would therefore catalyze cleavage of the N-acetylglucosamine residue at the reducing end of alpha 1-acid glycoprotein oligosaccharides only following removal of the linking Asn. The activity was then detected in lysosomal extracts by using intact asialo-biantennary oligosaccharides labeled with [3H] galactose or N-acetyl[14C]glucosamine residues as a substrate. To prevent simultaneous digestion of the material from its nonreducing end, beta-D-galactosidase in the enzyme extract was first inactivated with the irreversible active site-directed inhibitor, beta-D-galactopyranosylmethyl-p-nitrophenyltriazene. The observed di-N-acetylchitobiose cleaving activity worked optimally at pH 3.4 and was uniquely associated with the lysosomal fraction of the liver homogenate. The enzyme also cleaved triantennary chains and di-N-acetylchitobiose, but failed to hydrolyze substrates that had been reduced with NaBH4. The new glycosidase was well separated from N-acetyl-beta-D-glucosaminidase (assayed with p-nitrophenyl-beta-D-glucosaminide) by gel filtration chromatography and had an apparent molecular weight of 37,000. A similar enzyme that hydrolyzes di-N-acetylchitobiose had previously been found in extracts of human liver (Stirling, J. L. (1974) FEBS Lett. 39, 171-175).
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PMID:A di-N-acetylchitobiase activity is involved in the lysosomal catabolism of asparagine-linked glycoproteins in rat liver. 308 72

The kidneys of man, sheep, cattle and pig were all found to contain 1-aspartamido-beta-acetylglucosamine amidohydrolase activity. However, among these, only human kidney was found to contain endo-beta-N-acetylglucosaminidase activity. The absence of this enzyme in the kidneys of sheep and cattle explains why the oligosaccharides accumulated in, and excreted by, sheep and cattle afflicted with disorders of glycoprotein catabolism (i.e. alpha-mannosidosis and beta-mannosidosis) contain two N-acetylglucosamine residues at the reducing terminus instead of one, as is the case for human patients afflicted with similar disorders.
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PMID:Absence of endo-beta-N-acetylglucosaminidase activity in the kidneys of sheep, cattle and pig. 312 7

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

The intestinal brush-border enzyme sucrase-isomaltase splits sucrose into its component monosaccharides, glucose and fructose. A deficiency of the enzyme leads to sucrose intolerance. We studied the synthesis and intracellular processing of sucrase-isomaltase, using human intestinal explants in organ culture. Pulse-chase experiments with [35S]methionine followed by immunoprecipitation, sodium dodecyl sulfate-polyacrylamide-gel electrophoresis, and fluorography of labeled sucrase-isomaltase demonstrated that the molecule was initially recognized as a protein with a relative molecular weight (Mr) of 205,000. This was apparently converted to a species of 225,000 Mr within two hours. We studied the glycosylation of the protein using endo-beta-N-acetylglucosaminidase H and peptide-N4-(N-acetyl-beta-glucosaminyl)-asparagine amidase digestion of oligosaccharide side chains of the two forms of sucrase-isomaltase. The results showed that the early-appearing 205-kd (kilodalton) molecule contained high-mannose asparagine-linked oligosaccharides, and that the later-appearing, 225-kd molecule contained highly processed (mature) carbohydrate chains. Studies in a patient with primary sucrase-isomaltase deficiency demonstrated normal translation and high-mannose glycosylation of the precursor but a failure in further processing of the oligosaccharides, with subsequent intracellular degradation of the glycoprotein and undetectable enzymatic activity of intestinal sucrase. Abnormal intracellular processing of the enzyme was the probable mechanism of enzyme deficiency in this patient.
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PMID:A study of the molecular pathology of sucrase-isomaltase deficiency. A defect in the intracellular processing of the enzyme. 380 85

Echis carinatus venom was separated into twenty fractions by means of ultrafiltration and CM-Sephadex C-50 column chromatography. Fraction II possessed inhibitory activity on the aggregation of washed rabbit platelets and fraction XII possessed the procoagulant and platelet aggregation-inducing activity. Both were further purified by gel filtration on a Sephacryl S-200 column. The purified aggregation inducer was a glycoprotein with procoagulant activity 10-12-times that of the crude venom. It possessed proteinase and amidase but was devoid of esterase activity. The molecular weight was 16 000, and it contained 8.7% of neutral sugar. The isoelectric point was pH 7.6. The purified aggregation inhibitor was a single peptide chain with a molecular weight of 6800 and contained 22.1% of neutral sugar. The isoelectric point was pH 4.8. It was devoid of any enzymatic activity of the crude venom. The IC50 was about 10 micrograms/ml on the thrombin-induced platelet aggregation. The inhibitory activity was fully retained after the treatment of the venom aggregation inhibitor with neuraminidase, but was completely destroyed by sodium metaperiodate. Upon heat treatment at 90 degrees C, the venom aggregation inhibitor was heat stable at pH 5.5 for 4 h, but was completely destroyed after 2 h at pH 8.9 and retained about 50% of its inhibitory activity of the control at pH 7.2 for 4 h. The venom aggregation inhibitor decreased the elasticity of the whole blood clot, and this effect was related to its inhibitory action on platelet aggregation instead of blood coagulation.
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PMID:Characterization of the platelet aggregation inducer and inhibitor from Echis carinatus snake venom. 401 43

An enzyme was identified in human serum which unlike lysozyme cleaved the amide bond between N-acetyl-muramic acid and L-alanine of the peptide side chain of the rigid layer (murein) of Escherichia coli. The N-acetyl-muramyl-L-alanine amidase released all of the peptide side chains including those to which the lipoprotein is bound. A portion of the peptide side chains of the Micrococcus lysodeikticus murein was also hydrolysed from the polysaccharide chains. E. coli, M. lysodeikticus, Bacillus subtilis and Staphylococcus aureus were not killed by the amidase. Treatment of E. coli with EDTA or osmotic shock rendered the cells sensitive to the amidase and they were killed. Possible biological functions of the amidase are discussed. The enzyme was separated from lysozyme in human serum. Gel permeation chromatography indicated a molecular weight of the active enzyme of 82,000 while gel electrophoresis in the presence of sodium dodecyl sulfate revealed a molecular weight of 75,000. Thus, the enzyme probably consists of a single polypeptide chain. Incubation with neuraminidase rendered the amidase more basic suggesting the release of sialic acid residues. The modified glycoprotein disclosed an increased activity to murein. Enzyme activity was inhibited by p-chloromercuribenzene sulfonate and ethyleneglycol-bis(2-aminomethyl) tetraacetate (EGTA) at 1 and 0.2 mM concentration, respectively, whereas EDTA up to 5 mM was without effect. The amidase was also inactivated by agents that reduce disulfide bridges.
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PMID:Murein hydrolase (N-acetyl-muramyl-L-alanine amidase) in human serum. 615 47

We have studied the regional distribution of glycoasparagine storage material in the brain in aspartylglycosaminuria, a condition characterized by inherited deficiency of lysosomal N-aspartyl-beta-N-acetylglucosamine amidohydrolase. Gaschromatographic measurements of the main accumulating glycoprotein-derived metabolite, N-acetylglucosaminyl-asparagine (GlcNAc-Asn), in 12 defined cerebral areas showed that GlcNAc-Asn is rather evenly distributed in the brain. The mean concentrations ranged from 0.454 mg/g wet tissue (corpus callosum) to 0.0610 mg/g (pons). The GlcNAc-Asn concentrations tended to be higher in grey matter areas than in white matter areas. GlcNAc-Asn was identified in the isolated neuronal fraction, but not in the myelin fraction, by mass-fragmentographic techniques. Electron-microscopic reexamination of a brain biopsy specimen revealed, in addition to the abundant presence of storage lysosomes in the neuronal perikarya, numerous cytoplasmic inclusions in brain capillary endothelial cells and pericytes as well as in occasional macrophages. The results indicate that the glycoasparagine storage material is not limited to expected cortical areas in aspartylglycosaminuria, but is distributed in a rather constant fashion in all cerebral grey and white matter areas studied.
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PMID:Regional distribution of glycoasparagine storage material in the brain in aspartylglycosaminuria. 616 50

Vipera russellii venom was separated into thirteen fractions by means of DEAE-Sephadex A-50 column chromatography. Fraction III possessed anticoagulant and phospholipase A activities and Fraction XI possessed procoagulant and caseinolytic activities, both were further purified by gel filtration on Sephacryl S-200 column. Purified procoagulant (Component II) was a two-chain protein with molecular weight of 86 000 consisting of A-chain (Mr 66 000) and B-chain (Mr 20 000). It was a glycoprotein containing 7.8% neutral sugar and 715 amino-acid residues. The procoagulant activity was 10-times that of the crude venom. It was an acidic proteinase with isoelectric point of pH 4.2. Upon heat treatment at 60 degrees C, Component II was stable at pH 5.5 and 7.2 for 3 h, but was destroyed completely after 30 min at pH 8.9. It was devoid of esterase or amidase activity. Purified anticoagulant (Component I) was a single peptide chain with molecular weight of 16 000. It was carbohydrate free and contained 136 amino-acid residues. It was a basic protein with an isoelectric point of larger than pH 10. It was a potent phospholipase A with an enzymatic activity of 510 +/- 30 mumol/min per mg using phosphatidylcholine as substrate, and 1 microgram/ml was sufficient to cause 100% hemolysis by the indirect hemolytic method. Upon heat treatment at 90 degrees C, Component I was heat stable at pH 5.5 for more than 3 h, but was destroyed completely after 2 h at pH 7.2 and 8.9. The anticoagulant activity of Component I could be neutralized by platelet factor 3, tissue thromboplastin and cephalin.
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PMID:Purification and properties of the main coagulant and anticoagulant principles of Vipera russellii snake venom. 672 70


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