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)

The addition of nitrate, EDTA and dithiothreitol to the enzyme extraction buffer resulted in improved stability of the assimilatory nitrate reductase activity from the food yeast Candida utilis at both 4 degrees C and -10 degrees C. By incorporating this critical step in the following sequence the yeast NAD(P)H: nitrate oxidoreductase (EC 1.6.6.2) was purified approximately 68-fold by protamine sulphate precipitation, calcium gel adsorption, ion exchange chromatography and gel filtration. Both NADPH-nitrate reductase and NADH-nitrate reductase activities remained in constant association and ratio (2:3) during the entire course of purification. The enzyme showed an absolute requirement of NADPH or NADH for its activity. Maximal enzyme activity was obtained with 10-120 micrograms protein in a 10 min assay at 30 degrees C at pH 6.5, with an apparent Michaelis constant of 0.69 mM for nitrate as substrate. The enzyme is a molybdoflavo-protein involving sulphydryl groups, and is highly sensitive to free reducing agents, heavy metal ions and electron-transfer inhibitors. The results also suggested possible involvement of a second metal ion, perhaps iron, which was hypothesized to participate in the electron transfer scheme catalysed by this enzyme.
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PMID:Partial purification and properties of the assimilatory nitrate reductase of the food yeast Candida utilis. 378 22

The reduced nicotinamide adenine dinucleotide phosphate (NADPH)-nitrate oxidoreductase (EC 1.6.6.2) from Aspergillus nidulans was purified over 200-fold by use of salt fractionation, gel filtration, and ion-exchange chromatography. The purified enzyme was specific for NADPH and catalyzed reduction of nitrate, cytochrome c from isolated mitochondria of Aspergillus, and mammalian cytochrome c. An S(0.725) (20, w) of 7.8 was derived with sucrose density gradient centrifugation, and a Stokes radius of 6.4 nm was derived by gel filtration on Sephadex G-200. From these values, a molecular weight of 197,000 was computed, assuming v = 0.725 cm(3)/g. The spectral properties of the purified enzyme suggested a flavine component was present but revealed no pattern indicative of a hemoprotein. A cytochrome c, similar to the cytochrome c from isolated mitochondria, was found unassociated with the nitrate reductase after ion-exchange chromatography. No NADPH-nitrate reductase activity was detected in isolated mitochondria. Spectrally discernable reduction of the flavine component of the enzyme at 450 nm was noted after reaction with NADPH. This reduction was inhibited by p-chloromercuribenzoate but not by KCN. The addition of nitrate to NADPH reduced enzyme caused a reoxidation of the flavine component via a reaction which was inhibited by KCN but not by p-chloromercuribenzoate. The half-life of the purified enzyme at 37 C was 20 min for NADPH-nitrate reductase and 35 min for NADPH-cytochrome c reductase.
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PMID:Characterization of the reduced nicotinamide adenine dinucleotide phosphate-nitrate reductase of Aspergillus nidulans. 439 43

1. In rice seedlings synthesis of methyl viologen-nitrite reductase was stimulated by light, as was that of NADH-nitrate oxidoreductase (EC 1.6.6.1). A small residual effect of light on the synthesis of the enzymes persisted in the dark for a short time. 2. In etiolated seedlings exposed to light and nitrate, a lag period of 3h was necessary before enzyme synthesis commenced, whereas in green seedlings kept in the dark for 36h, synthesis of both the enzymes started as soon as light and nitrate were provided. 3. Experiments with cycloheximide suggested that fresh protein synthesis in light was necessary for formation of active enzymes. Mere activation by light of inactive enzymes or their precursors, was not involved. 4. In green seedlings synthesis of nitrite reductase was more sensitive to chloramphenicol than that of nitrate reductase. In chloramphenicol-treated etiolated seedlings, however, synthesis of both the enzymes was inhibited to the same extent on subsequent light-treatment. 5. A close correlation was observed between inhibition of the Hill reaction by 3-(3,4-dichlorophenyl)-1,1-dimethylurea and simazin [2-chloro-4,6-bis(ethylamino)-s-triazine] (at high concentration) and the inhibition of enzyme synthesis. At lower concentrations, however, simazin stimulated nitrate reductase. 6. In a single leaf synthesis of enzymes was observed only in portions exposed to light, whereas little activity was present in the dark covered part. 7. CO(2) deprivation severely inhibited the synthesis of enzymes in the light. Sucrose could not reverse this effect. 8. In excised embryos cultured in synthetic media containing sucrose, light was also essential for enzyme formation. 9. It is suggested that redox changes taking place in the green tissues as a result of the Hill reaction create conditions favourable for the induced synthesis of nitrate reductase and nitrite reductase.
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PMID:Role of light in the synthesis of nitrate reductase and nitrite reductase in rice seedlings. 466 75

In an earlier paper (Cove, 1966) it was reported that the kinetics of appearance of nitrate reductase (NADPH-nitrate oxidoreductase, EC 1.6.6.3) on the addition of nitrate to a growing culture of Aspergillus nidulans were different in certain respects from those found for many Escherichia coli enzymes. When urea is used as an initial nitrogen source, a further difference is found: enzyme synthesis is no longer continuous. This interruption of synthesis does not appear to be due to synchronous cell division in the culture, nor to be due to accumulation of ammonia. Fluctuations in the intracellular concentration of nitrate, though appearing to be partly responsible for the discontinuity of enzyme syntheses, cannot account for all the observations. Two related hypotheses are put forward to explain this discontinuity of synthesis; each suggests that nitrate reductase is intimately concerned with its own synthesis. One possibility is that the enzyme when it is not in the form of a complex with nitrate is a co-repressor of its own synthesis, and the other that the enzyme is its own repressor.
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PMID:Kinetic studies of the induction of nitrate reductase and cytochrome c reductase in the fungus Aspergillus nidulans. 604 55

In vitro complementation of the soluble assimilatory NAD(P)H-nitrate reductase (NAD(P)H:nitrate oxidoreductase, EC 1.6.6.2) was attained by mixing cell-free preparations of Chlamydomonas reinhardii mutant 104, uniquely possessing nitrate-inducible NAD(P)H-cytochrome c reductase, and mutant 305 which possesses solely the nitrate-inducible FMNH2- and reduced benzyl viologen-nitrate reductase activities. Full activity and integrity of NAD(P)H-cytochrome c reductase from mutant 104 and reduced benzyl viologen-nitrate reductase from mutant 305 are needed for the complementation to take place. A constitutive and heat-labile molybdenum-containing cofactor, that reconstitutes the NAD(P)H-nitrate reductase activity of nit-1 Neurospora crassa but is incapable of complementing with 104 from C. reinhardii, is present in the wild type and 305 algal strains. The complemented NAD(P)H-nitrate reductase has been purified 100-fold and was found to be similar to the wild enzyme in sucrose density sedimentation, molecular size, pH optimum, kinetic parameters, substrate affinity and sensitivity to inhibitors and temperature. From previous data and data presented in this article on 104 and 305 mutant activities, it is concluded that C. reinhardii NAD(P)H-nitrate reductase is a heteromultimeric complex consisting of, at least, two types of subunits separately responsible for the NAD(P)H-cytochrome c reductase and the reduced benzyl viologen-nitrate reductase activities.
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PMID:In vitro complementation of assimilatory NAD(P)H-nitrate reductase from mutants of Chlamydomonas reinhardii. 645 69

Assimilatory nitrate reductase (NAD(P)H-nitrate oxidoreductase, EC 1.6.6.2) from the green alga Ankistrodesmus braunii can be purified to homogeneity by dye-ligand chromatography on blue-Sepharose. The purified enzyme, whose turnover number is 623 s-1, presents an optimum pH of 7.5 and Km values of 13 microM, 23 microM and 0.15 mM for NADH, NADPH and nitrate, respectively. The NADH-nitrate reductase activity exhibits an iso ping pong bi bi kinetic mechanism. The molecular weight of the native nitrate reductase is 467 400, while that of its subunits is 58 750. These values suggest an octameric structure for the enzyme, which has been confirmed by electron microscopy. As deduced from spectrophotometric and fluorimetric studies, the enzyme contains FAD and cytochrome b-557 as prosthetic groups. FAD is not covalently bound to the protein and is easily dissociated in diluted solutions from the enzyme. Its apparent Km value is 4 nM, indicative of a high affinity of the enzyme for FAD. The results of the quantitative analyses of prosthetic groups indicate that nitrate reductase contains four molecules of flavin, four heme irons, and two atoms of molybdenum. The three components act sequentially transferring electrons from reduced pyridine nucleotides to nitrate, thus forming a short electron transport chain along the protein. A mechanism is proposed for the redox interconversion of the nitrate reductase activity. Inactivation seems to occur by formation of a stable complex of reduced enzyme with cyanide or superoxide, while reactivation is a consequence of reoxidation of the inactive enzyme. Both reactions imply the transfer of only one electron.
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PMID:Assimilatory nitrate reductase from the green alga Ankistrodesmus braunii. 668 79

The assimilatory NADPH-nitrate oxidoreductase (EC 1.6.6.3) from Aspergillus nidulans was purified by means of affinity chromatography and analyzed by agarose isoelectric focusing and two-dimensional electrophoresis. NADPH-nitrate reductase activity was not activated by oxidation with potassium ferricyanide and was irreversibly inhibited by acrylamide. Electrophoresis of nitrate reductase in 7% polyacrylamide gels resulted in rapid loss of enzyme activity. Isoelectric focusing of purified enzyme in agarose gels resulted in the homogeneous band that exhibited NADPH-nitrate reductase, NADPH-cytochrome c reductase and reduced methyl viologen-nitrate reductase activities, which corresponded to an isoelectric point of 6.12 +/- 0.05 at 22 degrees C. Two-dimensional electrophoresis of focused nitrate reductase on SDS-polyacrylanide gel slabs yielded a single subunit of 54000 molecular weight. Acid treatment of the enzyme and subsequent isoelectric focusing resulted in a protein with a strongly acidic isoelectric point and reduced methyl viologen-nitrate reductase activity. It released another protein with a strongly basic isoelectric point which was inactive. It is postulated that the overall association of flavoprotein protomers with both heme and cytochrome b1 components confers a small net negative charge upon the native heteromultimer and accounts for its slightly acidic isoelectric point.
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PMID:Isoelectric focusing and two-dimensional analysis of purified nitrate reductase from Aspergillus nidulans. 675 5

Nitrate reductase (NADPH:nitrate oxidoreductase; EC 1.6.6.1-3) was purified to apparent homogeneity from mycelium of Penicillium chrysogenum. The final preparation catalyzed the NADPH-dependent, FAD-mediated reduction of nitrate with a specific activity of 170-225 units X mg of protein-1. Gel filtration and glycerol density centrifugation yielded, respectively, a Stokes radius of 6.3 nm and an s20,w of 7.4. The molecular weight was calculated to be 199,000. On sodium dodecyl sulfate gels, the enzyme displayed two almost contiguous dye-staining bands corresponding to molecular weights of about 97,000 and 98,000. The enzyme prefers NADPH to NADH (kspec ratio = 2813), FAD to FMN (kspec ratio = 141), FAD (+ NADPH) to FADH2 (kspec ratio = 12,000), and nitrate to chlorate (kspec ratio = 4.33), where the kspec (the specificity constant for a given substrate) represents Vmax/Km. The Penicillium enzyme will also catalyze te NADPH-dependent, FAD-mediated reduction of cytochrome c with a specific activity of 647 units X mg of protein-1 (Kmcyt = 1.25 X 10(-5) M), and the reduced methyl viologen (MVH2, i.e. methyl viologen + dithionite)-dependent, NADPH and FAD-independent reduction of nitrate with a specific activity of 250 units X mg of protein-1 kmMVH2 = 3.5 X 10(-6) M). Initial velocity studies showed intersecting NADPH-FAD and nitrate-FAD reciprocal plot patterns. The NADPH-nitrate pattern was a series of parallel lines at saturating and unsaturating FAD levels. NADP+ was competitive with NADPH, uncompetitive with nitrate (at saturating and unsaturating FAD levels), and a mixed-type inhibitor with respect to FAD. Nitrite was competitive with nitrate, uncompetitive with NADPH (at saturating and unsaturating FAD levels), and a mixed-type inhibitor with respect to FAD. At unsaturating nitrate and FAD, NADPH exhibited substrate inhibition, perhaps as a result of binding to the FAD site(s). At very low FAD concentrations, low concentrations of NADP+ activated the reaction slightly. The initial velocity and product inhibition patterns are consistent with either of the two kinetic mechanisms. One (rather unlikely) mechanism involves the rapid equilibrium random binding of all ligands with (a) NADP+ and NADPH mutually exclusive, (b) nitrate and nitrite mutually exclusive, (c) the binding of NADPH strongly inhibiting the binding of nitrate and vice versa, (d) the binding of NADPH strongly promoting the binding of nitrite and vice versa, and (e) the binding of nitrate strongly promoting the binding of NADP+ and vice versa...
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PMID:Nitrate reductase from Penicillium chrysogenum. Purification and kinetic mechanism. 679 May 45

Gamma-irradiation induced high levels of nitrate reductase activity (NADH:nitrate oxidoreductase, EC 1.6.6.1) in callus of Haworthia mirabilis Haworth. Subcultures of gamma-irradiated tissues showed autonomous growth on minimal medium. We were able to mimic the effects of gamma-irradiation by inducing nitrate reductase activity in unirradiated callus with exogenous auxin and kinetin. These results revealed that induction of nitrate reductase activity by gamma-irradiation is mediated through in vitro activation of hormone synthesis in callus cells.
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PMID:Gamma-irradiation activates biochemical systems: induction of nitrate reductase activity in plant callus. 695 74

NADPH-nitrate reductase [NADPH : nitrate oxidoreductase, EC 1.6.6.3] was purified 500-fold from Aspergillus nidulans with an overall yield of about 20%. The purified enzyme catalyzed NADPH-nitrate, NADPH-cytochrome c, FADH2-nitrate and reduced methyl viologen-nitrate reductase activities. Its molecular weight was estimated to be 180,000 from the Stokes radius and sedimentation coefficient. The oxidized enzyme exhibited an absorption spectrum having a peak at 412 nm and a broad shoulder at about 450 nm. When reduced with NADPH, absorption peaks appeared at 423 (Soret), 527 (beta) and 557 (alpha) nm, and absorption in the 450 nm region decreased. Upon treatment of the reduced enzyme with KNO3, the spectrum returned to that of the oxidized enzyme. The presence of protoheme in the enzyme was confirmed by the absorption spectrum of reduced pyridine hemochromogen. It was concluded that a b-type cytochrome ("cytochrome b-557") is present in the enzyme and is involved in the intramolecular electron transport from NADPH to nitrate. The NADPH-nitrate and NADPH-cytochrome c reductase activities, but not the other two activities, were significantly decreased by incubation of the enzyme at 37 degrees C in the absence of FAD. Analysis by SDS slab gel electrophoresis suggested that the nitrate reductase consists of two each of two subunits of 59,000 and 38,000 daltons and that a dissociation of 38,000 subunits from the native enzyme occurs during heat treatment, resulting in alteration of the catalytic activity.
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PMID:Purification and characterization of the assimilatory NADPH-nitrate reductase of Aspergillus nidulans. 704 1


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