Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:1.7.1.2 (nitrate reductase)
 3,861 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The functional structure of assimilatory NADH-nitrate reductase from spinach leaves was studied by limited proteolysis experiments. After incubation of purified nitrate reductase with trypsin, two stable products of 59 and 45 kDa were observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The fragment of 45 kDa was purified by Blue Sepharose chromatography. NADH-ferricyanide reductase and NADH-cytochrome c reductase activities were associated with this 45-kDa fragment which contains FAD, heme, and NADH binding fragment. After incubation of purified nitrate reductase with Staphylococcus aureus V8 protease, two major peaks were observed by high performance liquid chromatography size exclusion gel filtration. FMNH2-nitrate reductase and reduced methyl viologen-nitrate reductase activities were associated with the first peak of 170 kDa which consists of two noncovalently associated (75-90-kDa) fragments. NADH-ferricyanide reductase activity, however, was associated with the second peak which consisted of FAD and NADH binding sites. Incubation of the 45-kDa fragment with S. aureus V8 protease produced two major fragments of 28 and 14 kDa which contained FAD and heme, respectively. These results indicate that the molybdenum, heme, and FAD components of spinach nitrate reductase are contained in distinct domains which are covalently linked by exposed hinge regions. The molybdenum domain appears to be important in the maintenance of subunit interactions in the enzyme complex.
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PMID:Limited proteolysis of the nitrate reductase from spinach leaves. 319 46

Dimethyl sulfoxide reductase, a terminal electron transfer enzyme, was purified from anaerobically grown Escherichia coli harboring a plasmid which codes for dimethyl sulfoxide reductase. The enzyme was purified to greater than 90% homogeneity from cell envelopes by a three-step purification procedure involving extraction with the detergent Triton X-100, chromatofocusing, and DEAE ion-exchange chromatography. The purified enzyme was composed of three subunits with molecular weights of 82,600, 23,600, and 22,700 as identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The native molecular weight was determined by gel electrophoresis to be 155,000. The purified enzyme contained 7.5 atoms of iron and 0.34 atom of molybdenum per mol of enzyme. The presence of molybdopterin cofactor in dimethyl sulfoxide reductase was identified by reconstitution of cofactor-deficient NADPH nitrate reductase activity from Neurospora crassa nit-I mutant and by UV absorption and fluorescence emission spectra. The enzyme displayed a very broad substrate specificity, reducing various N-oxide and sulfoxide compounds as well as chlorate and hydroxylamine.
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PMID:Purification and properties of Escherichia coli dimethyl sulfoxide reductase, an iron-sulfur molybdoenzyme with broad substrate specificity. 328 May 46

Biochemical and microbiological studies were conducted to characterize the mechanism of bacterial formation of N-nitrosomorpholine from morpholine and nitrite at neutral pH. Nitrosating activity was markedly induced when bacteria were cultured anaerobically in minimal culture medium containing nitrate, while the presence of cysteine or tungsten in the medium inhibited induction. Of various metals, coenzymes and inhibitors tested for their effects on in vitro nitrosation of morpholine, potassium cyanide, sodium azide, NAD(P)H and nitrate strongly inhibited nitrosation. Several mutants of Escherichia coli A10 strain were prepared in order to examine whether nitrosation activity is linked to specific loci. Niridazole-resistant mutants, which lack nitroreductase, had as much nitrosating activity as the original E. coli A10, but chlorate-resistant mutants had completely lost this activity. A good correlation was observed between nitrate reductase activity and nitrosating activity in these mutants. These results indicate that bacterial nitrosation is an enzyme-mediated reaction closely associated with molybdenoenzymes such as the nitrate reductase/formate hydrogenlyase system.
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PMID:Biochemical studies on the catalysis of nitrosation by bacteria. 330 Oct 45

Nitrate reductase (nitrite: (acceptor) oxidoreductase, EC 1.7.99.4) and trimethylamine N-oxide reductase (NADH : trimethylamine-N-oxide oxidoreductase, EC 1.6.6.9) activities were reconstituted by incubation of the association factor FA (the putative product of the chlB gene) with the soluble extract of the chlB mutant grown anaerobically in the presence of trimethylamine N-oxide. When soluble extracts of the chlB mutant grown on 10 mM sodium tungstate, a molybdenum competitor, were used in complementation systems, no enzymatic reactivation was observed. Heated extracts of the parental strain 541 were shown to contain a thermoresistant molybdenum cofactor by their ability to reactivate NADPH-nitrate reductase activity in the nit1 mutant of Neurospora crassa. By complementation of parental strain heated extract with association factor FA and soluble extract of the chlB mutant grown in the presence of sodium tungstate, we were able to show for the first time that the molybdenum cofactor is an activator common to the in vitro reconstitution of both nitrate reductase and trimethylamine-N-oxide reductase activities.
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PMID:Molybdenum cofactor: a compound in the in vitro activation of both nitrate reductase and trimethylamine-N-oxide reductase activities in Escherichia coli K12. 352 87

The chlorate-resistant (chlR) mutants are pleiotropically defective in molybdoenzyme activity. The inactive derivative of the molybdoenzyme, respiratory nitrate reductase, present in the cell-free extract of a chlB mutant, can be activated by the addition of protein FA, the probable active product of the chlB locus. Protein FA addition, however, cannot bring about the activation if 10 mM sodium tungstate is included in the culture medium for the chlB strain. The inclusion of a heat-treated preparation of a wild-type or chlB strain prepared after growth in the absence of tungstate, restores the protein-FA-dependent activation of nitrate reductase. All attempts to activate nitrate reductase in extracts prepared from tungstate-grown wild-type Escherichia coli strains failed. It appears that during growth with tungstate, the possession of the active chlB gene product leads to the synthesis of a nitrate reductase derivative which is distinct from that present in the tungstate-grown chlB mutant. Heat-treated preparations from chlA and chlE mutants which do not possess molybdenum cofactor activity fail to restore the activation. Fractionation by gel filtration of the heat-treated preparation from a wild-type strain produced two active peaks in the eluate of approximate Mr 12000 and less than or equal to 1500. The active material in the heat-treated extract was resistant to exposure to proteinases, but after such treatment the active component, previously of approximate Mr 12000, eluted from the gel filtration column with the material of Mr less than or equal to 1500. The active material is therefore of low molecular mass and can exist either in a protein-bound form or in an apparently free state. Molybdenum cofactor activity, assayed by the complementation of the apoprotein of NADPH:nitrate oxidoreductase in an extract of the nit-1 mutant of Neurospora crassa, gave a profile following gel filtration similar to that of the ability to restore respiratory nitrate reductase activity to the tungstate-grown chlB mutant soluble fraction. This was the case even after proteinase treatment of the heat-stable fraction. Analysis of the chlC (narC) mutant, defective in the structural gene for nitrate reductase, revealed that heat treatment is not necessary for the expression of the active component. Furthermore both the active component and molybdenum cofactor activity are present in corresponding bound and free fractions in the non-heat-treated soluble subcellular fraction.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Activation in vitro of respiratory nitrate reductase of Escherichia coli K12 grown in the presence of tungstate. Involvement of molybdenum cofactor. 352 61

Escherichia coli trimethylamine N-oxide (TMAO) reductase I, the major enzyme among inducible TMAO reductases, was purified to homogeneity by an improved method including heat treatment, ammonium sulfate precipitation, and chromatographies on Bio-Gel A-1.5m, DEAE-cellulose, and Reactive blue-agarose. The molecular weight was estimated by gel filtration to be approximately 200,000. A single subunit peptide with a molecular weight of 95,000 was found by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This enzyme contained 1.96 atoms of molybdenum, 0.96 atoms of iron, 1.52 atoms of zinc, and less than 0.4 atoms of acid-labile sulfur per molecular weight of 200,000. The absorption spectrum of the enzyme showed a peak at 278 nm and a shoulder at 288 nm, but no characteristic absorption was found from 350 to 700 nm. A fluorescent derivative of molybdenum cofactor was found when the enzyme was boiled with iodine in acidic solution; its fluorescence spectra were almost the same as those of the form A derivative of molybdopterin found in sulfite oxidase. The molybdenum cofactor released from heated TMAO reductase I reconstituted nitrate reductase in the extracts of Neurospora crassa mutant strain nit-1 lacking molybdenum cofactor. Thus, TMAO reductase I contains molybdopterin, which is a common constituent of some molybdenum-containing enzymes. Some kinetic properties were also determined.
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PMID:Further characterization of trimethylamine N-oxide reductase from Escherichia coli, a molybdoprotein. 352 39

Lysozyme digestion and sonication of sodium dodecyl sulfate (SDS)-purified Klebsiella aerogenes murein sacculi resulted in the quantitative release of both subunits of nitrate reductase, as well as a number of other cytoplasmic membrane polypeptides (5.2%, by weight, of the total membrane proteins). Similar results were obtained after lysozyme digestion of SDS-prepared peptidoglycan fragments, which excluded the phenomenon of simple trapping of the polypeptides by the surrounding peptidoglycan matrix. About 28% of membrane-bound nitrate reductase appears to be tightly associated with the peptidoglycan. Additional evidence for this association was demonstrated by positive immunogold labeling of SDS-murein sacculi and thin sections of plasmolyzed bacteria. Qualitative amino acid analysis of trypsin-treated sacculi, a tryptic product of holo-nitrate reductase, and amino- and carboxypeptidase digests of both nitrate reductase subunits indicated the possible existence of a terminal anchoring peptide containing the following amino acids: (Gly)n, Trp, Ser, Pro, Ile, Leu, Phe, Cys, Tyr, Asp, and Lys.
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PMID:Part of respiratory nitrate reductase of Klebsiella aerogenes is intimately associated with the peptidoglycan. 354 73

1. Groups of four conventional (CV) rats ate natural or purified diets either with or without 100 g fat/kg and drank 0.235 M-sodium nitrate. The fats tested were butterfat, coconut oil, olive oil, maize oil and safflower oil. 2. Decreased urinary excretion of N-nitrosoproline (NPRO) was observed in rats fed on fat-supplemented diets compared with those fed on low-fat diets, with butterfat having the greatest effect of the fats tested. 3. Reduced excretion of NPRO was not the result of inhibition of the intragastric N-nitrosation reaction or absorption of nitrosamine from the gastrointestinal tract. 4. The availability of nitrite in aqueous solution was decreased by the fat diets but the effect was similar in all the fats tested. 5. Nitrate reductase activity was present in the forestomach contents of CV rats at pH greater than 4 and was apparently inhibited by feeding a fat diet. No nitrate reductase activity was detected in stomach contents of germ-free rats. 6. Nitrate reductase activity in stomach and small intestinal tissue was not altered by feeding a fat diet. 7. It was concluded that nitrate reductase activity in stomach contents was of microbial origin and the decreased urinary excretion of NPRO on feeding the fat diets was mainly due to the inhibition of nitrate reductase activity in stomach contents.
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PMID:Dietary fat and N-nitrosation in the rat. 367 44

Molybdenum cofactor (mocofactor) is extracted efficiently, free of impurities and in high concentrations, by acid treatment of xanthine oxidase and subsequent incubation of the precipitate with phosphate buffer containing EDTA, molybdate and oxygen. It is suggested that cofactor is bound to the enzyme via hydrophobic forces as well as via an oxygen-sensitive mechanism. Upon extraction, the capability to complement the apo nitrate reductase of Neurospora crassa nit-1 can be conserved only in the total absence of oxygen. Cysteine and glutathione were shown to protect efficiently free mocofactor from oxidation. Two species of active mocofactor, probably a molybdoform and a demolybdoform, could be separated by means of reversed-phase HPLC with a mobile phase of 5 mM sodium citrate at a pH of 6.5. The mode of interaction between either of these species with thiol reagents is discussed.
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PMID:Extraction and purification of molybdenum cofactor from milk xanthine oxidase. 369 96

Nitrate reductase of Mitsuokella multiacidus (formerly Bacteroides multiacidus) was solublized from the membrane fraction with 1% sodium deoxycholate and purified 40-fold by immunoaffinity chromatography on the antibody-Affi-Gel 10 column. The preparation showed a major band (86% of total protein) with enzyme activity and a minor band on polyacrylamide gel after disc electrophoresis in the presence of 0.1% Triton X-100. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis gave a major band, the relative mobility of which corresponded to a molecular weight of 160,000, and two minor bands. The molecular weight of the enzyme was determined to be 160,000 by gel filtration on Bio-Gel A-1.5 m in the presence of 0.1% deoxycholate. Molybdenum cofactor was detected in the enzyme by fluorescence spectroscopy and by complementation of nitrate reductase from the nit-1 mutant of Neurospora crassa. The M. multiacidus enzyme catalyzed reduction of nitrate, chlorate, and bromate using methyl viologen as an electron donor. The maximal activity was found at pH 6.2-7.5 for nitrate reduction. Either methyl or benzyl viologen served well as the electron donor, but FAD, FMN, and horse heart cytochrome c were not effective. Ferredoxin from Clostridium pasteurianum supplied electron to the nitrate reductase. The purified enzyme had Km values of 0.13 mM, 0.12 mM, and 0.22 mM for nitrate, methyl viologen, and ferredoxin, respectively. The enzyme activity was inhibited by cyanide (85% at 1 mM), azide (88% at 0.1 mM), and thiocyanate (75% at 10 mM).
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PMID:Purification and properties of nitrate reductase from Mitsuokella multiacidus. 371 Oct 52


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