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

The stereospecificity of the hydrogen removal from reduced pyridine nucleotides catalyzed by nitrate reductase (NADH : nitrate oxidoreductase, EC 1.6.6.1, and NAD(P)H : nitrate oxidoreductase, EC 1.6.6.2) was investigated. A high degree of enzyme purification was required to obtain conclusive results. Improvements are described for the purification of nitrate reductase from Chlorella fusca and from spinach (Spinacea oleracea, L.) leaves. The latter enzyme is shown to contain a cytochrome. With highly purified nitrate reductase preparations from Cl. fusca, Neurospora crassa, Rhodotorula glutinis and spinach leaves the stereospecificity of the reaction was determined to be predominantly of the A-type in all cases.
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PMID:The stereospecificity of nitrate reductase for hydrogen removal from reduced pyridine nucleotides. 1 53

Partially purified soluble rat liver guanylate cyclase [GTP pyrophosphate-lyase (cyclizing), EC 4.6.1.2] was activated by superoxide dismutase (superoxide: superoxide oxidoreductase, EC 1.15.1.1). This activation was prevented with KCN or glutathione, inhibitors of superoxide dismutase. Guanylate cyclase preparations formed superoxide ion. Activation by superoxide dismutase was further enhanced by the addition of nitrate reductase. Although guanylate cyclase activity was much greater with Mn2+ than with Mg2+ as sole cation cofactor, activation with superoxide dismutase was not observed when Mn2+ was included in incubations. Catalase also decreased the activation induced with superoxide dismutase. Thus, activation required the formation of both superoxide ion and H2O2 in incubations. Activation of guanylate cyclase could not be achieved by the addition of H2O2 alone. Scavengers of hydroxyl radicals prevented the activation. It is proposed that superoxide ion and hydrogen peroxide can lead to the formation of hydroxyl radicals that activate guanylate cyclase. This mechanism of activation can explain numerous observations of altered guanylate cyclase activity and cyclic GMP accumulation in tissues with oxidizing and reducing agents. This mechanism will also permit physiological regulation of guanylate cyclase and cyclic GMP formation when there is altered redox or free radical formation in tissues in response to hormones, other agents, and processes.
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PMID:Activation of guanylate cyclase by superoxide dismutase and hydroxyl radical: a physiological regulator of guanosine 3',5'-monophosphate formation. 2 77

Neurospora crassa wild type STA4 NADPH-nitrate reductase (NADPH : nitrate oxidoreductase, EC 1.6.6.3) has been purified 5000-fold with an overall yield of 25--50%. The final purified enzyme contained 4 associated enzymatic activities: NADPH-nitrate reductase, FADH2-nitrate reductase, reduced methyl viologen-nitrate reductase and NADPH-cytochrome c reductase. Polyacrylamide gel electrophoresis yielded 1 major and 1 minor protein band and both bands exhibited NADPH-nitrate and reduced methyl viologen-nitrate reductase activities. SDS gel electrophoresis yielded 2 protein bands corresponding to molecular weights of 115 000 and 130 000. A single N-terminal amino acid (glutamic acid) was found and proteolytic mapping for the two separated subunits appeared similar. Purified NADPH-nitrate reductase contained 1 mol of molybdenum and 2 mol of cytochrome b557 per mol protein. Non-heme iron, zinc and copper were not detectable. It is proposed that the Neurospora assimilatory NADPH-nitrate reductase consists of 2 similar cytochrome b557-containing 4.5-S subunits linked together by one molybdenum cofactor. A revised electron flow scheme is presented. p-Hydroxymercuribenzoate inhibition was reversed by sulfhydryl reagents. Inhibitory pattern of p-hydroxymercuribenzoate and phenylglyoxal revealed accessible sulfhydryl and arginyl residue(s) as functional group(s) in the earlier part of electron transport chain as possibly the binding site of NADPH or FAD.
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PMID:Purification and characterization of homogeneous assimilatory reduced nicotinamide adenine dinucleotide phosphate-nitrate reductase from Neurospora crassa. 2 8

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

A molybdenum cofactor (Mo-co) from xanthine oxidase (xanthine:oxygen oxidoreductase, EC 1.2.3.2) can be isolated from the enzyme by a technique that has been used to isolate an iron-molybdenum cofactor (FeMo-co) from component I of nitrogenase. N-Methylformamide is used for the extraction of these molybdenum cofactors. Mo-co from xanthine oxidase activates nitrate reductase (NADPH:nitrate oxidoreductase, EC 1.6.6.2) in an extract from Neurospora crassa mutant strain Nit-1; however, FeMo-co is unable to activate nitrate reductase in strain Nit-1. Mo-co from xanthine oxidase is unable to activate nitrogenase in an extract of Azotobacter vinelandii mutant strain UW45. Inactive component I in this extract can be activated by FeMo-co. These results indicate that nitrate reductase and xanthine oxidase share a common molybdenum cofactor, but this cofactor is different from the molybdenum cofactor in nitrogenase.A. vinelandii synthesizes both Mo-co and FeMo-co. Mo-co is produced when the cells fix N(2) and also when they are repressed for nitrogenase synthesis by growth in a medium containing excess ammonium. However, FeMo-co is not produced when cells are grown in an ammonium-containing medium. Partially purified preparations of component I from A. vinelandii and Klebsiella pneumoniae contain both FeMo-co and Mo-co. The presence of both FeMo-co and Mo-co activities in partially purified preparations of component I explains previous reports of activation of inactive nitrate reductase in strain Nit-1 by acid-treated component I of nitrogenase. The Mo-co can be separated from FeMo-co in these preparations by chromatography on Sephadex G-100 in N-methylformamide. Both FeMo-co and Mo-co are sensitive to oxygen.
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PMID:Molybdenum cofactors from molybdoenzymes and in vitro reconstitution of nitrogenase and nitrate reductase. 14 98

Assimilatory nitrate reductase (EC 1.6.6.1 NADH:nitrate oxidoreductase) from Chlorella vulgaris purified by affinity chromatography was found to be homogeneous as judged by electrophoresis on sodium dodecyl sulfate-polyacrylamide gel and by analytical ultracentrifugal techniques. The molecular weight of the intact enzyme and that of the enzyme dissociated in 6 M GuHCl, determined by sedimentation equilibrium studies, were 280,000 +/- 10,000 and 90,000 +/- 5,000, respectively. Comparable values were obtained using the S20,w value and the D20,w values in Svedberg's equation. The D20,w values were determined by laser light-scattering measurements. Active enzyme centrifugation showed that the monomer is an active species. A quantitative re-evaluation of the prosthetic groups present (FAD, heme, and molybdenum) was also made and was consistent with the conclusion that the active monomer contains three subunits as previously deduced by Solomonson et al. ((1975) J. Biol. Chem. 250, 4120). Electron micrographs showed images which corresponded to three subunits, supporting the data obtained by hydrodynamic studies. The enzyme is not cigar-shaped, as previously surmised, but has a roughly globular structure.
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PMID:Physical studies on assimilatory nitrate reductase from Chlorella vulgaris. 50 Jun 68

A simple and rapid procedure to make yeast cells permeable by agitating with toluene-ethanol, (TE) 1:4, v/v was developed. The permeated cells retained their ability to catalyze certain enzyme reactions. Temperature and duration of agitation during TE treatment played an important role in retention of the catalytic potential of permeated cells. The in situ assay using permeated cell preparations was more sensitive even in the absence of added cofactors than in the vitro assay in detecting assimilatory nitrate reductase (NAD(P)H:nitrate oxidoreductase, EC 1.6.6.2) (NAR) activity in Candida utilis. Using in situ assay technique, different mechanisms regulating the biosynthesis of NAR in C. utilis were investigated. Nitrogen starvation did not lead to derepression of NAR. NO3-ions were absolutely essential for induction and maintenance of high levels of NAR activity. Cells grown on ammonium nitrate possessed relatively lower levels of NAR. Kinetics of NAR induction were followed as a function of time and inducer concentration. The influence of various cations on the induction of NAR by nitrate was investigated. A wide range of D-amino acids induced NAR synthesis. Of 22 L-amino acids tested only phenylalanine induced significant levels of NAR. Various intermediates of the pathway of nitrate reduction influenced the rate of NAR induction. There was a rapid disappearance of in vivo activity of the enzyme of induced yeast cells on nitrogen starvation, and the rate of loss was accelerated by the presence of NH4+.
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PMID:Regulatory properties of yeast nitrate reductase in situ. 94 16

Spinach (Spinacia oleracea L.) leaf nitrate reductase (NADH:NR;NADH:nitrate oxidoreductase, EC 1.6.6.1) activity was found to rapidly change during light/dark transitions. The most rapid and dramatic changes were found in a form of NR which was sensitive to inhibition by millimolar concentrations of magnesium. This form of NR predominated in leaves in the dark, but was almost completely absent from leaves incubated in the light for only 30 min. When the leaves were returned to darkness, the NR rapidly became sensitive to Mg2+ inhibition. Modulation of the overall reaction involving NADH as electron donor was also found when reduced methyl viologen was the donor (MV:NR), indicating that electron transfer had been blocked, at least in part, at or near the terminal molybdenum cofactor site. Changes in activity appear to be the result of a covalent modification that affects sensitivity of NR to inhibition by magnesium, and our results suggest that protein phosphorylation may be involved. NR was phosphorylated in vivo after feeding excised leaves [32P]Pi. The NR subunit was labeled exclusively on seryl residues in both light and dark. Tryptic peptide mapping indicated three major 32P-labeled phosphopeptide (Pp) fragments. Labeling of two of the P-peptides (designated Pp1 and 3) was generally correlated with NR activity assayed in the presence of Mg2+. In vivo, partial dephosphorylation of these sites (and activation of NR assayed with Mg2+) occurred in response to light or feeding mannose in darkness. The light effect was blocked completely by feeding okadaic acid via the transpiration stream, indicating the involvement of type 1 and/or type 2A protein phosphatases in vivo. While more detailed analysis is required to establish a causal link between the phosphorylation status of NR and sensitivity to Mg2+ inhibition, the current results are highly suggestive of one. Thus, in addition to the molecular genetic mechanisms regulating this key enzyme of nitrate assimilation, NR activity may be controlled in leaves by phosphorylation/dephosphorylation of the enzyme protein resulting from metabolic changes taking place during light/dark transitions.
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PMID:Reversible light/dark modulation of spinach leaf nitrate reductase activity involves protein phosphorylation. 160 45

A membrane-bound nitrate reductase (nitrite:(acceptor) oxidoreductase, EC 1.7.99.4) from the extremely halophilic bacterium Haloferax denitrificans was solubilized by incubating membranes in buffer lacking NaCl and purified by DEAE, hydroxylapatite, and Sepharose 6B gel filtration chromatography. The purified nitrate reductase reduced chlorate and was inhibited by azide and cyanide. Preincubating the enzyme with cyanide increased the extent of inhibition which in turn was intensified when dithionite was present. Although cyanide was a noncompetitive inhibitor with respect to nitrate, nitrate protected against inhibition. The enzyme, as isolated, was composed of two subunits (Mr 116,000 and 60,000) and behaved as a dimer during gel filtration (Mr 380,000). Unlike other halobacterial enzymes, this nitrate reductase was most active, as well as stable, in the absence of salt.
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PMID:Purification and properties of a dissimilatory nitrate reductase from Haloferax denitrificans. 165 31


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