Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:1.6.99.3 (diaphorase)
 5,903 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In vivo complementation between different wild and mutant strains defective for nitrate assimilation has been performed by isolating diploid strains from the appropriate crosses. Twenty-two diploids homozygous or heterozygous with respect to nitrate reduction and able to grow on nitrate medium were obtained and their diploid character demonstrated from analyses of mating type, cell volume, nuclear size and progeny of crosses with haploid wild-type. All diploids were assayed for overall- and terminal-nitrate reductase (NR) activity and for the occurrence of the NR-diaphorase subunit. Data on NR activities in heterozygotes carrying mutation(s) in structural gene(s) (nit-1 or nit-1a, nit-1b) agree with the heteromultimeric nature of the enzyme complex previously described (Franco et al. (1984) EMBO J 3: 1403-1407), and indicate that subunits are exchangeable to form hybrid enzymes. In addition, in vitro complementation tests with mutant nit-1 of C. reinhardtii indicate that this mutant has defective NR-diaphorase subunits but intact terminal subunits. Super-repression caused by the mutant allele nit-2 is suppressed by the wild allele in heterozygotes, which suggests a positive control by the nit-2 product on structural gene(s) transcription. Mutant alleles of genes for the biosynthesis of molybdenum-containing cofactor, either nit-4 or nit-5 and nit-6, were recessive in diploids carrying them. The mutant allele of nit-3, from strain 307, was codominant in all heterozygotes suggesting that nit-3 codes for a protein whose activity is limiting for the molybdenum-cofactor biosynthetic pathway.
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PMID:In vivo complementation analysis of nitrate reductase-deficient mutants in Chlamydomonas reinhardtii. 344 23

Two nitrate reductase (NaR)-deficient mutants of pea (Pisum sativum L.), E1 and A300, both disturbed in the molybdenum cofactor function and isolated, respectively, from cv Rondo and cv Juneau, were tested for allelism and were compared in biochemical and growth characteristics. The F1 plants of the cross E1 X A300 possessed NaR and xanthine dehydrogenase (XDH) activities comparable to those of the wild types, indicating that these mutants belong to different complementation groups, representing two different loci. Therefore, mutant E1 represents, besides mutant A300 and the allelic mutants A317 and A334, a third locus governing NaR and is assigned the gene destignation nar 3. In comparison with the wild types, cytochrome c reductase activity was increased in both mutants. The mutants had different cytochrome c reductase distribution patterns, indicating that mutant A300 could be disturbed in the ability to dimerize NaR apoprotein monomers, and mutant E1 in the catalytic function of the molybdenum cofactor. In growth characteristics studied, A300 did not differ from the wild types, whereas fully grown leaves of mutant E1 became necrotic in soil and in liquid media containing nitrate.
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PMID:Biochemical and genetic comparison of two nitrate reductase-deficient pea mutants disturbed in the cofactor. 347 18

1. Nitrate induces the development of NADH-nitrate reductase (EC 1.6.6.1), FMNH(2)-nitrate reductase and NADH-cytochrome c reductase activities in barley shoots. 2. Sucrose-density-gradient analysis shows one band of NADH-nitrate reductase (8S), one band of FMNH(2)-nitrate reductase activity (8S) and three bands of NADH-cytochrome c reductase activity (bottom layer, 8S and 3.7S). Both 8S and 3.7S NADH-cytochrome c reductase activities are inducible by nitrate, but the induction of the 8S band is much more marked. 3. The 8S NADH-cytochrome c reductase band co-sediments with both NADH-nitrate reductase activity and FMNH(2)-nitrate reductase activity. Nitrite reductase activity (4.6S) did not coincide with the activity of either the 8S or the 3.7S NADH-cytochrome c reductase. 4. FMNH(2)-nitrate reductase activity is more stable (t((1/2)) 12.5min) than either NADH-nitrate reductase activity (t((1/2)) 0.5min) or total NADH-cytochrome c reductase activity (t((1/2)) 1.5min) at 45 degrees C. 5. NADH-cytochrome c reductase and NADH-nitrate reductase activities are more sensitive to p-chloromercuribenzoate than is FMNH(2)-nitrate reductase activity. 6. Tungstate prevents the formation of NADH-nitrate reductase and FMNH(2)-nitrate reductase activities, but it causes superinduction of NADH-cytochrome c reductase activity. Molybdate overcomes the effects of tungstate. 7. The same three bands (bottom layer, 8S and 3.7S) of NADH-cytochrome c reductase activity are observed irrespective of whether induction is carried out in the presence or absence of tungstate, but only the activities in the 8S and 3.7S bands are increased. 8. The results support the idea that NADH-nitrate reductase, FMNH(2)-nitrate reductase and NADH-cytochrome c reductase are activities of the same enzyme complex, and that in the presence of tungstate the 8S enzyme complex is formed but is functional only with respect to NADH-cytochrome c reductase activity.
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PMID:Structural and functional relationships of enzyme activities induced by nitrate in barley. 432 54

Chlorella nitrate reductase catalyzes the reduction of nitrate to nitrite by NADH. Initial velocity studies showed that the kinetic mechanism is sequential, indicating that both substrates must bind to the enzyme before any products are released. Product inhibition with NAD and nitrite showed that competitive inhibition was observed when the inhibitor was similar to the varied substrate, while noncompetitive inhibition was observed when the inhibitor was dissimilar to the varied substrate. Likewise, dead-end inhibition with adenosine 5'-diphosphoribose and thiocyanate showed competitive inhibition when the inhibitor was similar to the varied substrate and noncompetitive inhibition when the inhibitor was dissimilar to the varied substrate. These results indicate that Chlorella nitrate reductase follows a random bi bi kinetic mechanism. Phosphate was found to stimulate NADH:nitrate reductase activity and 2-fold. The NADH:cytochrome c reductase activity associated with nitrate reductase was not affected by phosphate suggesting the effect of phosphate is on the nitrate-reducing moiety of the enzyme. Phosphate increases Vmax but has no effect on the apparent Km for nitrate.
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PMID:Kinetic mechanism of assimilatory NADH:nitrate reductase from Chlorella. 627 5

Experiments were performed to determine whether conditions which cause the rapid loss of nitrate reductase activity in Neurospora crassa mycelia were accompanied by the loss of antigenically detectable nitrate reductase protein. When mycelia with nitrate reductase activity were transferred to ammonia media, there was a rapid loss in the reduced nicotinamide adenine dinucleotide-nitrate reductase activity plus the parallel loss of the reduced nicotinamide adenine dinucleotide-diaphorase and the reduced methyl viologen-nitrate reductase activities associated with the nitrate reductase. In addition, there was the loss of cross-reacting material to anti-nitrate reductase antisera that was concomitant with the loss of nitrate reductase activity. When mycelia were exposed to either ammonia plus cycloheximide, nitrate plus cycloheximide, or nitrogen-free media, or to media which lacked an assimilable carbon source, the amount of cross-reacting material declined in concert with the nitrate reductase activity. The mutant nit-6, which lacks nitrite reductase activity, was exposed to ammonia or nitrate plus cycloheximide media. The nitrate reductase and the amount of cross-reacting material declined together as in the wild-type mycelia. We conclude that the loss of nitrate reductase activity was accompanied by the specific loss of this protein and that no pool of inactivated nitrate reductase molecules existed.
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PMID:Repression of nitrate reductase activity and loss of antigenically detectable protein in Neurospora crassa. 644 48

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

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

Six mutant strains (301, 102, 203, 104, 305, and 307) affected in their nitrate assimilation capability and their corresponding parental wild-type strains (6145c and 21gr) from Chlamydomonas reinhardii have been studied on different nitrogen sources with respect to NAD(P)H-nitrate reductase and its associated activities (NAD(P)H-cytochrome c reductase and reduced benzyl viologen-nitrate reductase) and to nitrite reductase activity. The mutant strains lack NAD(P)H-nitrate reductase activity in all the nitrogen sources. Mutants 301, 102, 104, and 307 have only NAD(P)H-cytochrome c reductase activity whereas mutant 305 solely has reduced benzyl viologen-nitrate reductase activity. Both activities are repressible by ammonia but, in contrast to the nitrate reductase complex of wild-type strains, require neither nitrate nor nitrite for their induction. Moreover, the enzyme from mutant 305 is always obtained in active form whereas nitrate reductase from wild-types needs to be reactivated previously with ferricyanide to be fully detected. Wild-type strains and mutants 301, 102, 104, and 307, when properly induced, exhibit an NAD(P)H-cytochrome c reductase distinguishable electrophoretically from constitutive diaphorases as a rapidly migrating band. Nitrite reductase from wild-type and mutant strains is also repressible by ammonia and does not require nitrate or nitrite for its synthesis. These facts are explained in terms of a regulation of nitrate reductase synthesis by the enzyme itself.
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PMID:Regulation of the nitrate-reducing system enzymes in wild-type and mutant strains of Chlamydomonas reinhardii. 681 63

Two fractions of nitrate reductase inhibitor activities were found in extracts of primary and regenerated roots of nitrate-grown rice seedlings. The inhibitor was proteinaceous in nature and specific to nitrate reductase. The main site of action of the inhibitor was the NADH: cytochrome c reductase component of nitrate reductase. NADH was able to protect the NADH:nitrate reductase against the inhibitor.
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PMID:Nitrate reductase inhibitor of rice plants. 718 23

Chlorella vulgaris was cultured on an ammonia-mineral salts medium until the nitrate reductase content reached a minimal level. These ammonia-grown cells were then induced by nitrate in the absence of molybdenum and of tungsten. A demolybdo nitrate reductase developed and reached high levels. This protein contained very little nitrate-reducing capacity, but had the full cytochrome c-reducing capacity of normal nitrate reductase. It was purified to homogeneity by the same procedures previously developed for the purification of nitrate reductase. The purified enzyme contained 1 molecule of heme and 1 molecule of FAD/subunit, but no detectable molybdenum or tungsten. This cytochrome c reductase was completely inhibited by antibodies raised against purified nitrate reductase of Chlorella. Mixtures prepared from normal nitrate reductase and the demolybdoenzyme could not be resolved by disc gel electrophoresis or by centrifugation in a density gradient. By a two-step enzyme induction (1, incubation with nitrate in absence of Mo; 2, incubation with Mo in absence of nitrate) the process of nitrate reductase synthesis could be cleanly separated from growth into two steps: Step 1, induction of cytochrome c reductase, was completely inhibited by cycloheximide. Step 2 was unaffected by cycloheximide, and most of the nitrate reductase synthesized accumulated in the form of the reversibly inactivated HCN complex of the enzyme.
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PMID:Purification and characterization of demolybdo nitrate reductase (NADH-cytochrome c oxidoreductase) of Chlorella vulgaris. 719 74


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