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:1.7.1.2 (nitrate reductase)
3,861 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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

The oxidation-reduction midpoint potential for the heme prosthetic group present in assimilatory nitrate reductase from Chlorella vulgaris has been determined by optical potentiometric titrations in the presence of dye mediators. At pH 7, the midpoint potential was determined to be -160 mV and corresponds to a reversible n = 1 redox process. The midpoint potential was unaltered by the use of NADH as reductant, unaffected by the presence of NAD+, cytochrome c, phosphate, cyanide, or alkaline pH. In addition, the redox potential of the heme was independent of modifications to the enzyme such as substitution of the molybdenum center with tungsten, or cleavage and separation of the enzyme into its flavin and heme/molybdenum domains. In contrast, the midpoint potential increased on decreasing the pH yielding a pH dependence of approximately 20 mV/pH unit within the range 5.5 to 7, suggesting the presence of a single, redox-associated, ionizable functional group on the protein with pKox = 5.8 and pKred = 6.1. At pH 7 and within the range 12 to 38 degrees C, the midpoint potential of the heme decreased by approximately 1 mV/degree. Values for delta S0 and delta H0 were calculated to be -25.6 e.u. and -4.0 kcal/mol.
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PMID:Thermodynamic properties of the heme prosthetic group in assimilatory nitrate reductase. 370 Mar 73

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

Nitrate uptake in Neurospora crassa has been investigated under various conditions of nitrogen nutrition by measuring the rate of disappearance of nitrate from the medium and by determining mycelial nitrate accumulation. The nitrate transport system is induced by either nitrate or nitrite, but is not present in mycelia grown on ammonia or Casamino Acids. The appearance of nitrate uptake activity is prevented by cycloheximide, puromycin, or 6-methyl purine. The induced nitrate transport system displays a K(m) for nitrate of 0.25 mM. Nitrate uptake is inhibited by metabolic poisons such as 2,4-dinitrophenol, cyanide, and antimycin A. Furthermore, mycelia can concentrate nitrate 50-fold. Ammonia and nitrite are non-competitive inhibitors with respect to nitrate, with K(i) values of 0.13 and 0.17 mM, respectively. Ammonia does not repress the formation of the nitrate transport system. In contrast, the nitrate uptake system is repressed by Casamino Acids. All amino acids individually prevent nitrate accumulation, with the exception of methionine, glutamine, and alanine. The influence of nitrate reduction and the nitrate reductase protein on nitrate transport was investigated in wild-type Neurospora lacking a functional nitrate reductase and in nitrate non-utilizing mutants, nit-1, nit-2, and nit-3. These mycelia contain an inducible nitrate transport system which displays the same characteristics as those found in the wild-type mycelia having the functional nitrate reductase. These findings suggest that nitrate transport is not dependent upon nitrate reduction and that these two processes are separate events in the assimilation of nitrate.
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PMID:Nitrate transport system in Neurospora crassa. 427 57

Downey, R. J. (University of Notre Dame, Notre Dame, Ind.). Nitrate reductase and respiratory adaptation in Bacillus stearothermophilus. J. Bacteriol. 91:634-641. 1966.-Bacillus stearothermophilus 2184 required nitrate to grow in the absence of oxygen. Like many facultative microorganisms, the growth obtained anaerobically was considerably less than that obtained aerobically, even though the dissimilatory reduction of nitrate is, in effect, anaerobic respiration. The ability to reduce nitrate depended on the induction of nitrate reductase. Although oxygen at low levels did not retard induction of the enzyme, enzyme synthesis was considerably lessened by aeration. A semisynthetic medium containing nitrate supported aerobic growth of the thermophile but did not support anaerobic growth. The adaptation to nitrate resulted in a decrease in the level of cytochrome oxidase normally present in aerobically grown cells. Although the aerobic oxidation of succinate by the respiratory enzymes from aerobically grown cells was inhibited by 2-N-heptyl-4-hydroxyquinoline-N-oxide, the anaerobic oxidation of succinate by nitrate in a similar preparation from nitrate-adapted cells was not. The nitrate reductase in the bacillus was strongly inhibited by cyanide and azide but not by carbon monoxide. The nitrate reductase catalyzed the anaerobic oxidation of reduced nicotinamide adenine dinucleotide, and appeared to transfer electrons from cytochrome b(1) to nitrate. Cytochrome c(1) did not appear to be involved in the transfer.
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PMID:Nitrate reductase and respiratory adaptation in Bacillus stearothermophilus. 428 85

Isolated membranes of Bacillus stearothermophilus 2184D can be disrupted by treatment with sodium dodecyl sulfate (SDS). This disruption is attended by a decreased turbidity of membrane suspensions and a differential loss of activities of the electron transport system. Reduced methyl viologen (MVH)-nitrate reductase activity is insensitive to SDS treatment, whereas reduced nicotinamide adenine dinucleotide (NADH)-nitrate reductase and cyanide-sensitive NADH oxidase activities are decreased by 80% at an SDS concentration of 0.5 mg/mg of membrane protein. NADH-menadione reductase activity is unaffected at this SDS concentration, but at higher detergent levels it also decreases in activity. The abilities of NADH to reduce and nitrate to oxidize the cytochrome components of the membrane were also decreased after SDS treatment. Dilution of solubilized membrane in buffer containing divalent cation results in formation of an aggregate with an increased turbidity and reconstituted NADH-nitrate reductase and cyanide-sensitive NADH oxidase activities. Of several cations tested, magnesium was the most effective, and the reconstitution process was pH-dependent with an optimum at pH 7.4. Intact and aggregated membranes had similar densities and cytochrome contents, and the sensitivity of NADH-nitrate reductase to several inhibitors was similar in intact and reconstituted membranes.
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PMID:Physical aggregation and functional reconstitution of solubilized membranes of Bacillus stearothermophilus. 433 10

Low concentrations (1-50mum) of ubiquinol(1) were rapidly oxidized by spheroplasts of Escherichia coli derepressed for synthesis of nitrate reductase using either nitrate or oxygen as electron acceptor. Oxidation of ubiquinol(1) drove an outward translocation of protons with a corrected -->H(+)/2e(-) stoichiometry [Scholes & Mitchell (1970) J. Bioenerg.1, 309-323] of 1.49 when nitrate was the acceptor and 2.28 when oxygen was the acceptor. Proton translocation driven by the oxidation of added ubiquinol(1) was also observed in spheroplasts from a double quinone-deficient mutant strain AN384 (ubiA(-)menA(-)), whereas a haem-deficient mutant, strain A1004a, did not oxidize ubiquinol(1). Proton translocation was not observed if either the protonophore carbonyl cyanide m-chlorophenylhydrazone or the respiratory inhibitor 2-n-heptyl-4-hydroxyquinoline N-oxide was present. When spheroplasts oxidized Diquat radical (DQ(+)) to the oxidized species (DQ(++)) with nitrate as acceptor, nitrate was reduced to nitrite according to the reaction: [Formula: see text] and nitrite was further reduced in the reaction: [Formula: see text] Nitrite reductase activity (2) was inhibited by CO, leaving nitrate reductase activity (1) unaffected. Benzyl Viologen radical (BV(+)) is able to cross the cytoplasmic membrane and is oxidized directly by nitrate reductase to the divalent cation, BV(++). In the presence of CO, this reaction consumes two protons: [Formula: see text] The consumption of these protons could not be detected by a pH electrode in the extra-cellular bulk phase of a suspension of spheroplasts unless the cytoplasmic membrane was made permeable to protons by the addition of nigericin or tetrachlorosalicylanilide. It is concluded that the protons of eqn. (3) are consumed at the cytoplasmic aspect of the cytoplasmic membrane. Diquat radical, reduced N-methylphenazonium methosulphate and its sulphonated analogue N-methylphenazonium-3-sulphonate (PMSH) and ubiquinol(1) are all oxidized by nitrate reductase via a haem-dependent, endogenous quinone-independent, 2-n-heptyl-4-hydroxyquinoline N-oxide-sensitive pathway. Approximate-->H(+)/2e(-) stoichiometries were zero with Diquat radical, an electron donor, 1.0 with reduced N-methylphenazonium methosulphate or its sulphonated analogue, both hydride donors, and 2.0 with ubiquinol(1) (QH(2)), a hydrogen donor. It is concluded that the protons appearing in the medium are derived from the reductant and the observed-->H(+)/2e(-) stoichiometries are accounted for by the following reactions occurring at the periplasmic aspect of the cytoplasmic membrane.: [Formula: see text]
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PMID:The mechanism of proton translocation driven by the respiratory nitrate reductase complex of Escherichia coli. 625 43

The assimilatory nitrate reductase from Chlorella contains flavin, heme, and molybdenum as prosthetic groups. The molybdenum in assimilatory nitrate reductase is associated with a pterin moiety (molybdopterin) as evidenced by the ability of the enzyme to donate active molybdenum cofactor to the Neurospora nitrate reductase mutant nit-1 and by the oxidative conversion of the pterin to two well characterized fluorescent derivatives. The properties of the molybdenum center have been examined by EPR spectroscopy. A molybdenum V signal, absent in the resting enzyme, is elicited upon reduction with NADH and abolished upon reoxidation with nitrate. Reaction of the reduced enzyme with cyanide also abolishes the molybdenum V signal. The line shape and g values of the signal show pH dependence analogous to those observed previously with hepatic sulfite oxidase. The gav for molybdenum V at pH 7.0 was 1.977 and at pH 9.0, 1.961. The signal observed at pH 7.0 exhibits interaction with a single exchangeable proton. Potentiometric titration of the molybdenum center at pH 7.0 indicates that the oxidation-reduction potentials of the molybdenum VI/V and molybdenum V/IV couples are -34 and -54 mV, respectively. These potentials are significantly different from the potentials of the molybdenum center of respiratory-type nitrate reductase and in fact quite closely resemble those of hepatic sulfite oxidase. The oxidized enzyme exhibits the EPR signal of a low spin ferric heme which is abolished upon reduction with NADH.
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PMID:Electron paramagnetic resonance studies on the molybdenum center of assimilatory NADH:nitrate reductase from Chlorella vulgaris. 631 88

In Aspergillus nidulans, chlorate strongly inhibited net nitrate uptake, a process separate and distinct from, but dependent upon, the nitrate reductase reaction. Uptake was inhibited by uncouplers, indicating that a proton gradient across the plasma membrane is required. Cyanide, azide, and N-ethylmaleimide were also potent inhibitors of uptake, but these compounds also inhibited nitrate reductase. The net uptake kinetics were problematic, presumably due to the presence of more than one uptake system and the dependence on nitrate reduction, but an apparent Km of 200 microM was estimated. In uptake assays, the crnA1 mutation reduced nitrate uptake severalfold in conidiospores and young mycelia but had no effect in older mycelia. Several growth tests also indicate that crnA1 reduces nitrate uptake. crnA expression was subject to control by the positive-acting regulatory gene areA, mediating nitrogen metabolite repression, but was not under the control of the positive-acting regulatory gene nirA, mediating nitrate induction.
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PMID:Nitrate uptake in Aspergillus nidulans and involvement of the third gene of the nitrate assimilation gene cluster. 635 Feb 63

The molecular basis for the action of two natural inactivator proteins, isolated from rice and corn, on a purified assimilatory nitrate reductase has been examined by several physical techniques. Incubation of purified Chlorella nitrate reductase with either rice inactivator protein or corn inactivator protein results in a loss of NADH:nitrate reductase and the associated partial activity, NADH:cytochrome c reductase, but no loss in nitrate-reducing activity with reduced methyl viologen as the electron donor. The molecular weight of the reduced methyl viologen:nitrate reductase species, determined by sedimentation equilibrium in the Beckman airfuge after complete inactivation with rice inactivator protein or with corn inactivator protein, was 595,000 and 283,000, respectively, compared to a molecular weight of 376,000 for the untreated control determined under the same conditions. Two protein peaks were observed after molecular-sieve chromatography on Sephacryl S-300 of nitrate reductase inactivated by corn inactivator protein. The Stokes radii of these fragments were 68 and 24 A, compared to a value of 81 A for untreated nitrate reductase. The large fragment contained molybdenum and heme but no flavin, and had nitrate-reducing activity with reduced methyl viologen as electron donor. The small fragment contained FAD but had no NADH:cytochrome c reductase or nitrate-reducing activities. Molecular weights determined by sodium dodecyl sulfate-gel electrophoresis were 67,000 and 28,000 for the large and small fragments, respectively, compared to a subunit molecular weight of 99,000 determined for the untreated control. No change in subunit molecular weight of nitrate reductase after inactivation by rice inactivator protein was observed. These results indicate that rice inactivator protein acts by binding to nitrate reductase. The stoichiometry of binding is 1-2 molecules of rice inactivator protein to one tetrameric molecule of nitrate reductase. Corn inactivator protein, in contrast, acts by cleavage of a Mr 30,000 fragment from nitrate reductase which is associated with FAD. The remaining fragment is a tetramer of Mr 70,000 subunits which retains nitrate-reducing activity and contains molybdenum and heme but has no NADH:dehydrogenase activity. The action of rice inactivator protein was partially prevented by NADH and completely prevented by a combination of NADH and cyanide, while the action of corn inactivator protein was not significantly affected by these effectors.
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PMID:Mode of action of natural inactivator proteins from corn and rice on a purified assimilatory nitrate reductase. 654 59


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