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)

Peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F(PNGase F) from Flavobacterium meningosepticum is a highly useful enzyme for the structural analysis of N (asparagine)-linked carbohydrate chains derived from glycoproteins. The enzyme was enriched using a published procedure [Tarentino AL, Gomez CM, Plummer TH, Jr (1984) Biochemistry 1985:4665-71; Tarentino AL, Plummer TH, Jr (1987) Methods Enzymol 138:770-78] and further purified by hydrophobic interaction HPLC on a weak hydrophobic TSK-Ether column from which it was eluted by a decreasing gradient of 1.7 M ammonium sulphate in 100 mM sodium phosphate, pH 7.0, containing 5 mM EDTA. To determine the optimal conditions for a complete deglycosylation of glycoproteins by PNGase F, experiments were performed with human alpha 1-acid glycoprotein, because the five complex type carbohydrate chains are quite resistant to enzymic hydrolysis. The influence of different detergents on the enzyme reaction was studied. Complete deglycosylation of human alpha 1-acid glycoprotein was achieved by the use of 60 mU/ml PNGase F in 0.25 M sodium phosphate buffer, pH 8.6, containing 0.2% (w/v) SDS, 20 mM mercaptoethanol and 0.5% Mega-10.
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PMID:Optimized deglycosylation of glycoproteins by peptide-N4-(N-acetyl-beta-glucosaminyl)-asparagine amidase from Flavobacterium meningosepticum. 213 46

A protease that nicks the approximately 150-kilodalton (kDa) single-chain type A botulinum neurotoxin into the approximately 150-kDa di-chain form in vitro was isolated from Clostridium botulinum type A (Hall strain) cultures. The di-chain neurotoxin generated in vitro is composed of an approximately 50-kDa light chain and an approximately 100-kDa heavy chain which are disulfide linked and is indistinguishable from the di-chain neurotoxin that forms in vivo and is routinely isolated (M.L. Dekleva and B.R. DasGupta, Biochem. Biophys. Res. Commun. 162:767-772, 1989). This enzyme was purified greater than 1,000-fold by ammonium sulfate precipitation, QAE-Sephadex Q-50, Sephadex G-100, and CM-Sephadex C-50 chromatography steps with the synthetic substrate N-benzoyl-DL-arginine-p-nitroanilide. The approximately 62-kDa amidase (protease) is a complex of 15.5- and 48-kDa polypeptides (determined by polyacrylamide gel electrophoresis) that could not be separated without sodium dodecyl sulfate. The enzyme has an isoelectric point of pH 5.73, a pH optimum of 6.2 to 6.4, an absolute requirement for a thiol-reducing agent as well as a divalent metallic cation (probably Ca2+) for activity, and a temperature optimum of 70 degrees C. Tests with several synthetic substrates indicated the high specificity of the enzyme for arginyl amide bonds.
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PMID:Purification and characterization of a protease from Clostridium botulinum type A that nicks single-chain type A botulinum neurotoxin into the di-chain form. 218 24

N-Glycosidase F (peptide-N4-(N-acetyl-beta-glycosaminyl)asparagine amidase; EC 3.5.1.52) catalyzes the cleavage of N-glycosidically linked carbohydrate chains between N-acetylglucosamine and asparagine. The structural gene was isolated by screening a Flavobacterium meningosepticum genomic DNA library in lambda gt10 with oligonucleotides, deduced from partial amino acid sequences of the protein. A clone with an open reading frame of 1062 bases was obtained. The amino acid sequence reveals a 42-residue-long leader peptide, which shows similarities to the endoglycosidase H-leader with respect to the cleavage site of the signal peptide, but is distinct from the ones known from other Gram-positive or -negative bacteria. The molecular weight of the native protein, derived from the DNA sequence, is in agreement with the molecular weight of the purified protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (35,000). Escherichia coli, transformed with a plasmid containing this DNA sequence, expresses N-glycosidase F activity. The enzyme with its natural Flavobacterium promoter and leader peptide is not secreted in E. coli but seems to be associated with cell membranes.
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PMID:Molecular cloning and heterologous expression of N-glycosidase F from Flavobacterium meningosepticum. 220 81

Methemoglobinemia produced by exposure to the herbicide propanil (3,4-dichloropropionanilide) is thought to be mediated by toxic metabolites formed during the hepatic clearance of the parent compound. We examined the metabolism of propanil and 3,4-dichloroaniline in rat liver microsomes to identify metabolites that may be involved in propanil-induced methemoglobinemia. The major pathway of propanil metabolism in microsomal incubations was acylamidase-catalyzed hydrolysis to 3,4-dichloroaniline. The reaction did not require NADPH, and was inhibited by the acylamidase inhibitors paraoxon and sodium fluoride. Oxidized metabolites were isolated by high-performance liquid chromatography, and identified as 2'-hydroxypropanil and 6-hydroxypropanil by comparison of their mass and nuclear magnetic resonance spectra to those of synthetic standards. Major microsomal metabolites of 3,4-dichloroaniline were 6-hydroxy-3,4-dichloroaniline and N-hydroxy-3,4-dichloroaniline. Both N-hydroxy-3,4-dichloroaniline and 6-hydroxy-3,4-dichloroaniline directly oxidized hemoglobin in rat erythrocyte suspensions in a concentration-dependent manner; however, the potency of N-hydroxy-3,4-dichloroaniline was at least an order of magnitude greater than that of 6-hydroxy-3,4-dichloroaniline.
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PMID:Metabolism of the arylamide herbicide propanil. I. Microsomal metabolism and in vitro methemoglobinemia. 231 34

Guinea pig alpha-macroglobulin was purified to apparent homogeneity by sequential chromatography on Sephacryl S-300, DEAE-cellulose, and hydroxyapatite. A molecular weight of 780,000 was obtained by equilibrium sedimentation. The preparation migrated as a single band of Mr = 180,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. Rabbit antiserum raised against the final preparation partially cross-reacted with human and rat alpha-2-macroglobulins but not with rat alpha-1-macroglobulin. Guinea pig alpha-macroglobulin stimulated the amidolytic activity of trypsin towards a small substrate, but inhibited the proteolytic activity of trypsin towards remazol brilliant blue hide powder. When treated with trypsin or methylamine, four thiol groups per molecule were newly generated. The reaction with trypsin proceeded with at least at two different rates: half of the thiol groups were generated in a fast reaction and the remaining half in a slower reaction. On the other hand, such a two-step reaction was not detected in the reaction with methylamine. The methylamine-treated alpha-macroglobulin retained half the capacity to bind trypsin and its mobility in polyacrylamide gel under nondenaturing conditions remained virtually unchanged. These properties are in marked contrast to those reported for human alpha-2-macroglobulin, but resemble those of rat alpha-2- and mouse alpha-macroglobulins. The amidase activity of trypsin bound to guinea pig alpha-macroglobulin was impaired by soybean trypsin inhibitor to a much greater degree than that of trypsin bound to human or rat alpha-2-macroglobulin.
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PMID:Isolation and characterization of alpha-macroglobulin from guinea pig plasma. 242 4

Large scale purification of human active urinary kallikrein is described. The final preparation was found homogeneous by means of SDS Page electrophoresis, amino acid composition and N-terminal analysis. The apparent molecular weight, determined on SDS Page electrophoresis, was 4.4 X 10(4). Comparative inhibition studies of the kininogenase and the amidase activities pointed out differences in the sensitivity of these two activities. Sodium inhibited amidase activity whereas kininogenase activity required the presence of this cation. In contrast, kininogenase activity was more sensitive to cadmium inhibition than amidase activity. Antibody against purified kallikrein did not completely inhibit amidase activity in crude urine. These discrepancies are consistent with the existence of several amidase activities in urine and also with possibly distinct catalytic sites on the same molecule, accordingly consideration of the methodology used appears very important when comparing results from different studies.
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PMID:Purification of human active urinary kallikrein: comparative inhibition studies of kininogenase and amidolytic activities. 250 80

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

Penicillin amidase was coupled to a periodate-oxidised dextran by reductive alkylation in the presence of sodium cyanoborohydride. A loss of activity (25%) was observed but the conjugate enzyme dextran was more thermostable than the native enzyme. Native and dextran-conjugated penicillin amidase were immobilised on amino activated silica (Promaxon, Spherosil, Aerosil) by a classical method using glutaraldehyde for the native enzyme and reductive alkylation for the modified enzyme. Good relative activity of the enzymes was obtained after insolubilisation. Immobilisation of both native and modified enzymes resulted in the thermostabilisation of the penicillin amidase.
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PMID:Stabilisation and immobilisation of penicillin amidase. 268 13

XAC (xanthine amine congener, 8-[4-[(2-aminoethyl)-aminocarbonylmethyloxy]phenyl]-1,3-dipropy lxanthine is a potent adenosine antagonist that reverses the reduction in urine flow, sodium excretion and heart rate produced by the adenosine agonist, N6-cyclohexyladenosine. New derivatives of XAC in which the primary amino group has been condensed to the gamma-carboxyl group of glutamic acid have been synthesized as prodrugs. These amino acid-XAC conjugates, which are considerably less potent than XAC in competitive binding assays at A1-adenosine receptors, are designed for selective enzymatic activation in the kidneys. The gamma-glutamyl xanthine derivatives are substrates for gamma-glutamyl transferase (EC 2.3.2.2) to generate an amine-functionalized xanthine. N-acetyl-gamma-L-glutamyl-XAC is not active in vivo, consistent with inability of renal acylase (EC 3.5.1.14) to hydrolyze the acetyl group, a prerequisite step for the production of XAC from this molecule. The xanthine derivatives, gamma-L-glutamyl-XAC and gamma-L-glutamyl-gamma-L-glutamyl-XAC are metabolized to XAC and produce a diuresis in vivo.
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PMID:Adenosine receptor prodrugs: towards kidney-selective dialkylxanthines. 274 13

The acyl coenzyme A (CoA):6-aminopenicillanic acid (6-APA) acyltransferase of Penicillium chrysogenum AS-P-78 was purified to homogeneity, as concluded by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing. The enzyme is a monomer with a molecular weight of 30,000 +/- 1,000 and a pI of about 5.5. The optimal pH and temperature were 8.0 and 25 degrees C, respectively. This enzyme converts 6-APA into penicillin by using phenylacetyl CoA or phenoxyacetyl CoA as acyl donors. The pure enzyme showed a high specificity and affinity for 6-APA and did not accept benzylpenicillin, 7-aminocephalosporanic acid, cephalosporin C, or isocephalosporin C as substrates. The enzyme converted isopenicillin N into penicillin G, although with a lower efficiency than when 6-APA was used as the substrate. It did not show penicillin G acylase activity. The acyl CoA:6-APA acyltransferase required dithiothreitol or other thiol-containing compounds, and it was protected by thiol-containing reagents against thermal inactivation. The acyltransferase was inhibited by several divalent and trivalent cations and by p-chloromercuribenzoate and N-ethylmaleimide. The activity was absent in four different mutants that were blocked in penicillin biosynthesis.
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PMID:Purification to homogeneity and characterization of acyl coenzyme A:6-aminopenicillanic acid acyltransferase of Penicillium chrysogenum. 282 13


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