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 assimilatory NADPH-nitrate reductase (NADPH:nitrate oxidoreductase, EC 1.6.6.3) from Neurospora crassa is competitively inhibited by 3-aminopyridine adenine dinucleotide (AAD) and 3-aminopyridine adenine dinucleotide phosphate (AADP) which are structural analogs of NAD and NADP, respectively. The amino group of the pyridine ring of AAD(P) can react with nitrous acid to yield the diazonium derivative which may covalently bind at the NAD(P) site. As a result of covalent attachment, diazotized AAD(P) causes time-dependent irreversible inactivation of nitrate reductase. However, only the NADPH-dependent activities of the nitrate reductase, i.e. the overall NADPH-nitrate reductase and the NADPH-cytochrome c reductase activities, are inactivated. The reduced methyl viologen- and reduced FAD-nitrate reductase activities which do not utilize NADPH are not inhibited. This inactivation by diazotized AADP is prevented by 1 mM NADP. The inclusion of 1 muM FAD can also prevent inactivation, but the FAD effect differs from the NADP protection in that even after removal of the exogenous FAD by extensive dialysis or Sephadex G-25 filtration chromatography, the enzyme is still protected against inactivation. The FAD-generated protected form of nitrate reductase could again be inactivated if the enzyme was treated with NADPH, dialyzed to remove the NADPH, and then exposed to diazotized AADP. When NADP was substituted for NADPH in this experiment, the enzyme remained in the FAD-protected state. Difference spectra of the inactivated nitrate reductase demonstrated the presence of bound AADP, and titration of the sulfhydryl groups of the inactivated enzyme revealed that a loss of accessible sulfhydryls had occurred. The hypothesis generated by these experiments is that diazotized AADP binds at the NADPH site on nitrate reductase and reacts with a functional sulfhydryl at the site. FAD protects the enzyme against inactivation by modifying the sulfhydryl. Since NADPH reverses this protection, it appears the modifications occurring are oxidation-reduction reactions. On the basis of these results, the physiological electron flow in the nitrate reductase is postulated to be from NADPH via sulfhydryls to FAD and then the remainder of the electron carriers as follows: NADPH leads to -SH leads to FAD leads to cytochrome b-557 leads to Mo leads to NO-3.
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PMID:Reactions of the Neurospora crassa nitrate reductase with NAD(P) analogs. 1 30

Induced wildtype cells of A. nidulans rapidly lost NADPH--linked nitrate reductase activity when subjected to carbon and or nitrogen starvation. A constitutive mutant at the regulatory gene for nitrate reductase, nir Ac 1, rapidly lost nitrate reductase activity upon carbon starvation. This loss of activity is thought to be due to a decrease in the NADPH concentration in the cells. Cell free extracts from wildtype cells grown in the presence of nitrate, rapidly lost their nitrate reductase activity when incubated at 25 degrees C. NADPH prevented this loss of activity. Wildtype cells grown in the presence of nitrate and urea have a higher initial NADPH:NADP+ ratio and cell free extracts from such cells lost their nitrate reductase activity slower than extracts of cells grown with nitrate alone. The Pentose Phosphate Pathway mutant, pppB-1, had a lower NADPH concentration compared with the wildtype grown under the same conditions and cell free extracts lost their nitrate reductase activity more rapidly than the wildtype. Cell free extracts of nirAc-1 and a non-inducible mutant for nitrate reductase, nirA- -14, upon incubation lost little of their nitrate reductase activity.
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PMID:In vivo and in vitro studies of nitrate reductase regulation in Asperillus nidulans. 1 26

The reduced nicotinamide adenine dinucleotide phosphate (NADPH):nitrate oxidoreductase (EC 1.6.6.2) from Aspergillus nidulans wild-type bi-1 was purified by means of salt fractionation, gel filtration, affinity chromatography, and polyacrylamide gel electrophoresis. Enzyme which was adsorbed on Cibacron blue agarose could be eluted with 2 mM NADPH or 2 mM oxidized NADP (NADP(+)), the former being about three times more effective than the latter. About half the total NADPH:nitrate reductase activity adsorbed on agarose required elution with 1 M NaCl. This salt-elutable form remained active with NADPH and was not converted to the NADPH-elutable form after readsorption on Cibacron blue agarose. The NADPH-eluted enzyme exhibited a markedly different electrophoretic mobility than the enzyme eluted with NADP(+) or NaCl. After electrophoresis on polyacrylamide gels, the NADPH-eluted NADPH:nitrate reductase was separated into four proteins, two of which contained nonheme iron and exhibited reduced methyl viologen-nitrate reductase activity. None of these proteins, singly or in combination, reduced nitrate with NADPH as substrate. Difference spectra analyses and specific heme iron stains revealed the presence of cytochrome b(557) in the largest of the proteins. The molecular weights of the four proteins, which were determined from the relationship of their mobilities on varied concentrations of acrylamide gel, were 360,000, 300,000, 240,000, and 118,000. The subunit molecular weights of these, which are determined via sodium dodecyl sulfate slab gel electrophoresis, were 49,000, 50,000, and 75,000. The key role of NADPH in maintenance of the active form of the heteromultimer is further substantiated.
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PMID:Further characterization of the reduced nicotinamide adenine dinucleotide phosphate: nitrate oxidoreductase in Aspergillus nidulans. 3 44

Nicotinamide adenine dinucleotide phosphate (reduced form)-nitrate reductase was freed from ammonium repression in a Neurospora crassa mutant having drastically lowered glutamine synthetase activity, gln-1a. The general phenomenon of nitrogen metabolite repression required glutamine or some aspect of glutamine metabolism.
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PMID:Nitrogen metabolite repression of nitrate reductase in Neurospora crassa: effect of the gln-1a locus. 3 43

Nucleotide sequences were determined for cDNA clones for squash NADH:nitrate oxidoreductase (EC 1.6.6.1), which is one of the most completely characterized forms of this higher plant enzyme. An open reading frame of 2754 nucleotides began at the first ATG. The deduced amino acid sequence contains 918 residues, with a predicted Mr = 103,376. The amino acid sequence is very similar to sequences deduced for other higher plant nitrate reductases. The squash sequence has significant similarity to the amino acid sequences of sulfite oxidase, cytochrome b5, and NADH:cytochrome b5 reductase. Alignment of these sequences with that of squash defines domains of nitrate reductase that appear to bind its 3 prosthetic groups (molybdopterin, heme-iron, and FAD). The amino acid sequence of the FAD domain of squash nitrate reductase was aligned with FAD domain sequences of other NADH:nitrate reductases, NADH:cytochrome b5 reductases, NADPH:nitrate reductases, ferredoxin:NADP+ reductases, NADPH:cytochrome P-450 reductases, NADPH:sulfite reductase flavoproteins, and Bacillus megaterium cytochrome P-450BM-3. In this multiple alignment, 14 amino acid residues are invariant, which suggests these proteins are members of a family of flavoenzymes. Secondary structure elements of the structural model of spinach ferredoxin:NADP+ reductase were used to predict the secondary structure of squash nitrate reductase and the other related flavoenzymes in this family. We suggest that this family of flavoenzymes, nearly all of which reduce a hemoprotein, be called "flavoprotein pyridine nucleotide cytochrome reductases."
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PMID:The sequence of squash NADH:nitrate reductase and its relationship to the sequences of other flavoprotein oxidoreductases. A family of flavoprotein pyridine nucleotide cytochrome reductases. 174 31

l-Glutamate uptake, thiourea uptake, and methylammonium uptake and the intracellular ammonium concentration were measured in wild-type and mutant cells of Aspergillus nidulans held in various concentrations of ammonium and urea. The levels of l-glutamate uptake, thiourea uptake, nitrate reductase, and hypoxanthine dehydrogenase activity are determined by the extracellular ammonium concentration. The level of methylammonium uptake is determined by the intracellular ammonium concentration. The uptake and enzyme characteristics of the ammonium-derepressed mutants, meaA8, meaB6, DER3, amrA1, xprD1, and gdhA1, are described. The gdhA mutants lack normal nicotinamide adenine dinucleotide phosphate-glutamate dehydrogenase (NADP-GDH) activity and are derepressed with respect to both external and internal ammonium. The other mutant classes are derepressed only with respect to external ammonium. The mutants meaA8, DER3, amrA1, and xprD1 have low levels of one or more of the l-glutamate, thiourea, and methylammonium uptake systems. A model for ammonium regulation in A. nidulans is put forward which suggests: (i) NADP-GDH located in the cell membrane complexes with extracellular ammonium. This first regulatory complex determines the level of l-glutamate uptake, thiourea uptake, nitrate reductase, and xanthine dehydrogenase by repression or inhibition, or both. (ii) NADP-GDH also complexes with intracellular ammonium. This second and different form of regulatory complex determines the level of methylammonium uptake by repression or inhibition, or both.
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PMID:Ammonium regulation in Aspergillus nidulans. 414 65

Nicotinamide adenine dinucleotide phosphate, reduced form (NADPH)-nitrate reductase and its related enzyme activities, NADPH-cytochrome c reductase and reduced benzyl viologen-nitrate reductase, are all induced following the transfer of ammonia-grown wild-type Neurospora mycelia to nitrate medium. After nitrate reductase is induced to the maximal level, the addition of an ammonium salt to, or the removal of nitrate from, the cultures results in a rapid inactivation of nitrate reductase and its two partial component activities. This rapid inactivation is slowed down by the protein synthesis inhibitor, cycloheximide. Experiments on the mixing of extracts in vitro rule out the presence of an inhibitor of nitrate reductase in free form in extracts containing inactivated nitrate reductase. Ammonia does not inhibit the uptake of nitrate by the mycelia. Inactivation of nitrate reductase in vivo by ammonia depends on the concentration of the ammonium salt and is not reversed by increasing the nitrate concentration of the medium. The nitrate-inducible NADPH-cytochrome c reductase activity and reduced benzyl viologen-nitrate reductase activity respectively of the nitrate-nonutilizing mutants nit-1 and nit-3 are not inactivated in vivo by the addition of an ammonium salt or the withdrawal of nitrate. This finding suggests that the integrity of the nitrate reductase complex is required for the in vivo inactivation of nitrate reductase and its associated activities.
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PMID:Regulation of nitrate reductase in Neurospora crassa: stability in vivo. 440 13

Growth of Neurospora crassa on media containing NH4+ leads to the repression of a variety of permeases and alternative pathways which would generate NH4+, so called "ammonium repression." The mutant am2 which lacks NADP-GDH is not subject to ammonium repression of nitrate reductase or urea permease, but like the wild type has repressed levels of these systems when grown in the presence of proline, glutamate or glutamine. The glutamine synthetase (GS) mutant gln-1a has derepressed levels of the aforementioned systems unless grown with glutamine. The oligomeric state of GS depends upon the nitrogen sufficiency of the cell, a tetrameric form predominates under conditions of nitrogen limitation and an octameric form under conditions of nitrogen sufficiency. We have found that the tetrameric form GS predominates in the mutants am2 and gln-1a when they are ammonium derepressed. Th mechanism of NH4+ repression in N. crassa is thought to entail a cessation of positive gene action by the product of the nit-2 regulatory gene. We propose that under conditions of NH4+ sufficiency, and hence glutamine sufficiency, the octameric form of GS represses nit-2 gene expression and thereby achieves ammonium repression.
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PMID:The role fo glutamine synthetase and glutamine metabolism in nitrogen metabolite repression, a regulatory phenomenon in the lower eukaryote Neurospora crassa. 610 28

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

An assay for the simultaneous measurement of nitrite and nitrate, products of nitric oxide metabolism, is described. Others have reported pretreating sample by using nitrate reductase (NR) and NADPH to reduce endogenous NO3- before assaying the resultant NO2- using the Griess reaction. However, we found that the NADP+ formed during pretreatment interfered with the Griess reaction when NADPH was used at concentrations necessary to drive the NR reaction. For instance, 500 microM NADP+ in 100 microM NaNO3- (without NR) causes a 90% interference with the formation of Griess reaction product. To limit interference, we modified the method by decreasing the NADPH concentration to 1 microM. NADPH was regenerated by coupling the NR reaction with that catalyzed by glucose-6-phosphate dehydrogenase (GD). Using this method, NaNO3- standard curves were linear up to 100 microM and coincided with control curves obtained using NaNO2- incubated in parallel. Addition of urine up to a strength of 20% did not interfere with the assay. Comparison with an alternative assay based on cadmium reduction resulted in the following linear regression: [Cd method] = 0.915*[NR-GD method] + 0.37, r2 = 0.997. Coupling GD to NR to recycle NADPH allows this cofactor to be used at a low concentration so that interference with the Griess reaction is negligible.
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PMID:Sample pretreatment with nitrate reductase and glucose-6-phosphate dehydrogenase quantitatively reduces nitrate while avoiding interference by NADP+ when the Griess reaction is used to assay for nitrite. 773 51


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