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 reduction of nitrate by reduced nicotinamide-adenine dinucleotides, catalysed by extract of Candida utilis, exhibits an apparent high degree of stereospecificity for the 'B' methylene hydrogen atom of NADPH and mixed stereospecificity for the methylene hydrogen atoms of NADH. Purified nitrate reductase, on the other hand, exhibits 'A' stereospecificity for NADH and NADPH. The apparent switch of stereospecificity from the 'B' to the 'A' side of NADPH, which occurs after purification of the enzyme, is partly explained by the fact that in crude extracts nitrate is reduced completely to ammonia. Nitrite does not accumulate but is reduced to ammonia by nitrite dehydrogenase, which is 'B'-specific, so that up to 75% of hydrogen removed from NADPH during the reduction of nitrate could occur from the 'B' side. A further increase in the removal of hydrogen from the 'B' side of NADPH could be the kinetic isotope effect that is observed when ['A'-3H]NADPH is the reductant, the H--C bond being cleaved 2.3 times faster than the 3H--C bond. The mixed stereospecificity observed with NADH has been traced to an uncharacterized enzyme that catalyses a 'B'-specific exchange between NAD+ and NADH. This reaction is discussed in relation to the possibility that it may explain other cases of apparent mixed stereospecificity that have been reported.
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PMID:The stereospecificity of the reduction of nitrate by reduced nicotinamide-adenine dinucleotides catalysed by Candida utilis preparations. 689 Aug 12

Nitrite found in human saliva is the product of the microbial reduction of nitrate circulating through the salivary glands. Saliva samples were collected under controlled conditions from volunteers who were habitual users of different types of tobacco products (tobacco chewers, cigarette smokers, bidi smokers and masheri users) and from controls. The saliva samples were analysed for nitrite levels and for nitrate reductase activity spectrophotometrically. Samples were collected from two different areas, Bombay (urban) and Ghodegaon (rural). Salivary nitrite levels in the control groups ranged from 1.6 ppm in Ghodegaon to 11 ppm in Bombay. The nitrite levels of the masheri-using groups from both locations and of the tobacco-chewing group from Bombay were significantly higher than those of the controls. A number of volunteers showed undetectable levels of salivary nitrate reductase. Volunteers who chewed tobacco or used masheri had higher levels of nitrate reductase activity than the controls. However, there did not seem to be any clear correlation between nitrite levels and nitrate reductase activity in the saliva.
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PMID:Nitrate reductase activity and nitrite levels in the saliva of habitual users of various tobacco products. 720 53

The increasing importance of nitric oxide synthase has been underscored by the elucidation of its role in a growing number of normal and pathophysiological processes. Therefore, techniques for detection of nitrite/nitrate, oxidation products of the enzymatic conversion of arginine to citrulline and nitric oxide, should serve as useful tools in defining the contribution of NO synthase to these processes. We have developed a rapid and sensitive fluorometric assay for quantification of nitrite/nitrate based upon the reaction of nitrite with 2,3-diaminonaphthalene to form the fluorescent product, 1-(H)-naphthotriazole. The assay can be used to detect 10 nM nitrite, making it 50-100 times more sensitive than the well-known Griess assay. Moreover, the assay is adaptable to a 96-well plate format, facilitating the handling of a large number of samples including conditioned media from cell culture or the nitrite generated by the purified enzyme. Nitrite/nitrate levels in blood can also be monitored using this assay when it is combined with a filtration step (to remove hemoglobin) followed by conversion of the nitrate to nitrite by nitrate reductase. Thus, this fluorometric method combines speed and sensitivity with the handling of a large number of samples for the quantification of nitrite generated from in vivo and in vitro sources.
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PMID:A fluorometric assay for the measurement of nitrite in biological samples. 750 9

Nitrate uptake and its regulation were investigated using an ion-specific nitrate electrode for denitrifying Flexibacter canadensis under anaerobic conditions. Glucose supported a greater rate of nitrate uptake than did glycerol, glutamate, lactose, cellobiose, or ethanol. Nitrate uptake closely approximated Michaelis--Menten kinetics; the estimated Ks(glucose) and apparent Km(nitrate) for nitrate uptake were 21 and 44 microM, respectively. Nitrate disappearance was correlated with nitrite accumulation, and nitrate had an inhibitory effect on nitrite reduction. Oxygen inhibition of nitrate uptake increased as the percent air saturation increased, and reversed readily as the percent air saturation decreased. The minimal air saturation showing inhibition of nitrate uptake was about 2-4%. Azide and cyanide completely inhibited nitrate uptake. No nitrate uptake was observed in cells grown in the presence of 1 or 5 mM tungstate (no added molybdate). When molybdate (100-200 microM) was present in the medium, nitrate uptake was exhibited by organisms grown with 1 mM, but not with 5 mM, tungstate, indicating that nitrate uptake was dependent on the presence of an active nitrate reductase, and that competition between tungsten and molybdenum occurred during the formation of nitrate reductase. Nitrite production from nitrate by whole cells but not cell-free extracts was inhibited by 2,4-dinitrophenol and carbonyl cyanide m-chlorophenylhydrazone, indicating that nitrate and (or) nitrite transport depended upon the electrochemical proton gradient.
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PMID:Cellular regulation of nitrate uptake in denitrifying Flexibacter canadensis. 807 52

An assay based on the oxidation of NADPH during the enzymatic conversion of nitrate to nitrate by Aspergillus nitrate reductase [EC 1.6.6.2] was developed for specific quantification of nitrate. This spectrophotometric method was used to measure nitrate present in human urine, human serum, and tissue culture medium. Used as a kinetic assay, the method exhibited (1) linearity over a range of 1.25 to 40 microM nitrate, (2) an upper sensitivity of 20 microM, (3) a lower sensitivity of 1.25 microM nitrate, and (4) intraday and interday variability ranging from 0.6 to 6.1%. To judge the acceptability of this method as a kinetic assay, we determined the Km for Aspergillus nitrate reductase to be 199 microM. The Km was based on analyzing three separate lots of commercially purified enzyme. Mean nitrate content of eight urine specimens analyzed by this assay (1111 microM) was not significantly different from that determined by a chemiluminescence method (1144 microM). Analysis of serum using the two methods showed mean nitrate concentrations of 23 and 36 microM, respectively. Based on serial dilutions of serum, the lower nitrate content of serum observed with nitrate reductase assay could not be explained by the presence of inhibitors. Rat pulmonary alveolar macrophages were induced to produce nitric oxide which oxidizes to nitrite and nitrate. Nitrite and nitrate present in tissue culture medium of unactivated and activated macrophages were in proportion to total nitrogen oxides (NO(x)) determined by the chemiluminescence method. We conclude that the Aspergillus nitrate reductase assay is an accurate spectrophotometric method for determining nitrate content of human urine and tissue culture supernatants.
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PMID:A spectrophotometric assay for nitrate using NADPH oxidation by Aspergillus nitrate reductase. 821 77

Nitrite functioned as an effective inducer of nitrate reductase, the enzyme responsible for the reduction of nitrate in the nitrate assimilation pathway in Candida utilis. Nitrite-induced synthesis of nitrate reductase in C. utilis was repressed by various metabolites of nitrate, including ammonia. Readily-assimilable sources of nitrogen such as ammonia and glutamate exerted a stronger repression on nitrate reductase induction than did less-readily assimilable hydrazine and hydroxylamine. Nitrite-mediated induction of nitrate reductase appeared more sensitive to repression by nitrate metabolites than was nitrate-mediated induction. Based on the inducer-specific differences in the sensitivity of the enzyme to repression by various intermediary metabolites and on other properties, it is proposed that the C. utilis nitrate reductase is either polymorphic or utilizes alternative receptor(s) for binding various gratuitous inducers including nitrite in initiating the induction pathway.
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PMID:Product-mediated regulation reveals the polymorphic nature of the yeast assimilatory nitrate reductase. 833 54

Eleven green individuals were isolated when 95000 M2 plants of barley (Hordeum vulgare L.), mutagenised with azide in the M1, were screened for nitrite accumulation in their leaves after nitrate treatment in the light. The selected plants were maintained in aerated liquid culture solution containing glutamine as sole nitrogen source. Not all plants survived to flowering and some others that did were not fertile. One of the selected plants, STA3999, from the cultivar Tweed could be crossed to the wild-type cultivar and analysis of the F2 progeny showed that leaf nitrite accumulation was due to a recessive mutation in a single nuclear gene, which has been designated Nir1. The homozygous nir1 mutant could be maintained to flowering in liquid culture with either glutamine or ammonium as sole nitrogen source, but died within 14 days after transfer to compost. The nitrite reductase cross-reacting material seen in nitrate-treated wild-type plants could not be detected in either the leaf or the root of the homozygous nir1 mutant. Nitrite reductase activity, measured with dithionite-reduced methyl viologen as electron donor, of the nitrate-treated homozygous nir1 mutant was much reduced but NADH-nitrate reductase activity was elevated compared to wild-type plants. We conclude that the Nir1 locus determines the formation of nitrite reductase apoprotein in both the leaf and root of barley and speculate that it represents either the nitrite reductase apoprotein gene locus or, less likely, a regulatory locus whose product is required for the synthesis of nitrite reductase, but not nitrate reductase.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:nir1, a conditional-lethal mutation in barley causing a defect in nitrite reduction. 843 74

Staphylococcus carnosus reduces nitrate to ammonia in two steps. (i) Nitrate was taken up and reduced to nitrite, and nitrite was subsequently excreted. (ii) After depletion of nitrate, the accumulated nitrite was imported and reduced to ammonia, which again accumulated in the medium. The localization, energy gain, and induction of the nitrate and nitrite reductases in S. carnosus were characterized. Nitrate reductase seems to be a membrane-bound enzyme involved in respiratory energy conservation, whereas nitrite reductase seems to be a cytosolic enzyme involved in NADH reoxidation. Syntheses of both enzymes are inhibited by oxygen and induced to greater or lesser degrees by nitrate or nitrite, respectively. In whole cells, nitrite reduction is inhibited by nitrate and also by high concentrations of nitrite (> or = 10 mM). Nitrite did not influence nitrate reduction. Two possible mechanisms for the inhibition of nitrite reduction by nitrate that are not mutually exclusive are discussed. (i) Competition for NADH nitrate reductase is expected to oxidize the bulk of the NADH because of its higher specific activity. (ii) The high rate of nitrate reduction could lead to an internal accumulation of nitrite, possibly the result of a less efficient nitrite reduction or export. So far, we have no evidence for the presence of other dissimilatory or assimilatory nitrate or nitrite reductases in S. carnosus.
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PMID:Physiology and interaction of nitrate and nitrite reduction in Staphylococcus carnosus. 860 76

The genes that encode the hc-type nitric-oxide reductase from Paracoccus denitrificans have been identified. They are part of a cluster of six genes (norCBQDEF) and are found near the gene cluster that encodes the cd1-type nitrite reductase, which was identified earlier [de Boer, A. P. N., Reijnders, W. N. M., Kuenen, J. G., Stouthamer, A. H. & van Spanning, R. J. M. (1994) Isolation, sequencing and mutational analysis of a gene cluster involved in nitrite reduction in Paracoccus denitrificans, Antonie Leeu wenhoek 66, 111-127]. norC and norB encode the cytochrome-c-containing subunit II and cytochrome b-containing subunit I of nitric-oxide reductase (NO reductase), respectively. norQ encodes a protein with an ATP-binding motif and has high similarity to NirQ from Pseudomonas stutzeri and Pseudomonas aeruginosa and CbbQ from Pseudomonas hydrogenothermophila. norE encodes a protein with five putative transmembrane alpha-helices and has similarity to CoxIII, the third subunit of the aa3-type cytochrome-c oxidases. norF encodes a small protein with two putative transmembrane alpha-helices. Mutagenesis of norC, norB, norQ and norD resulted in cells unable to grow anaerobically. Nitrite reductase and NO reductase (with succinate or ascorbate as substrates) and nitrous oxide reductase (with succinate as substrate) activities were not detected in these mutant strains. Nitrite extrusion was detected in the medium, indicating that nitrate reductase was active. The norQ and norD mutant strains retained about 16% and 23% of the wild-type level of NorC, respectively. The norE and norF mutant strains had specific growth rates and NorC contents similar to those of the wild-type strain, but had reduced NOR and NIR activities, indicating that their gene products are involved in regulation of enzyme activity. Mutant strains containing the norCBQDEF region on the broad-host-range vector pEG400 were able to grow anaerobically, although at a lower specific growth rate and with lower NOR activity compared with the wild-type strain.
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PMID:Mutational analysis of the nor gene cluster which encodes nitric-oxide reductase from Paracoccus denitrificans. 902 86

Nitrite and nitrate (NO2 and NO3), the oxidative products of nitric oxide (NO), were elevated in the plasma of rabbits on the third day following ligation of a coronary artery. This elevation coincided with increased activity of the inducible form of nitric oxide synthase (iNOS) in infarcted heart muscle. Data are reported which relate the elevated plasma concentrations of NO2+NO3 (NO(x)) to the increased induction of iNOS in an infarcted heart. NO2 and NO3 in plasma were measured by chemiluminescence. Nitrate was converted to nitrite by nitrate reductase. Plasma from the ear vein, right and left ventricle, and coronary sinus were analyzed for NO(x), and iNOS activity was enzymatically determined in infarcted, risk, and normal areas of the heart. The production equivalent of NO(x) by the heart and lung was also calculated. In addition, the effect of a specific inhibitor of iNOS, S-methylisothiourea sulfate (SMT) on plasma concentration and myocardial production of NO(x) was determined. It was concluded that the elevation of plasma NO(x) following onset of myocardial ischemia was directly related to increased induction of iNOS in the heart. This conclusion was based on a proportional and simultaneous increase in NO(x) plasma concentration with myocardial iNOS activation. The inhibitory effect of SMT furnished additional confirmation of the relationship between myocardial iNOS activation and NO(x) plasma levels in experimental myocardial infarction.
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PMID:Oxidation products of nitric oxide, NO2 and NO3, in plasma after experimental myocardial infarction. 904 16


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