Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:Q8NEX9 (reductase)
 26,410 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Woodward's reagent K (N-ethyl-5-phenylisoxazolium-3'-sulfonate) inactivated both soluble and membrane bound-ferredoxin-NADP+ reductase of spinach chloroplasts. Either NADP+ or NADPh afforded complete protection against modification. Ki and the apparent Kd for protection afforded by NADP+ depended on the ionic strength of the medium. Nucleophylic displacement of reagent bound to the soluble enzyme by [14C]glycine ethyl ester showed that 5 to 6 carboxyl groups/flavin were modified when the diaphorase activity was completely inhibited. In differential labeling experiments using NADP+ as protective agent, it was shown that enzyme inactivation was due to blocking of only 1 carboxyl group/mol. Derivatized reductase did not bind pyridine nucleotides. Protection by NADP+ of the membrane-bound reductase was higher, and the apparent Kd for NADP+ lower, in the light than in the dark. Inactivation increased abruptly with the external pH, indicating a progressive exposure of the carboxyl group as the pH was raised. The results presented suggest (a) the existence of a light-driven conformational change and a pH-dependent transition in membrane-bound ferredoxin-NADP+ reductase; (b) the presence of an essential carboxyl residue in the nucleotide binding site of the reductase.
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PMID:An essential carboxyl group at the nucleotide binding site of ferredoxin-NADP+ oxidoreductase. 689 98

A series of straight chain N-alkymaleimides was shown to simultaneously inactivate the reductase, transhydrogenase and diaphorase activities of yeast glutathione reductase (NAD(P)H: oxidized-glutathione oxidoreductase, EC 1.6.4.2.) at pH 7.5 and 25 degrees C. Apparent second-order rate constants for the inactivation of all enzyme activities exhibited parallel increases with increasing chainlength from C-2 through C-7 of the alkyl substituent of the enhanced binding of maleimides through nonpolar interactions with the enzyme. Reduction of the active site disulfide with NADPH was required prior to addition of maleimide for inactivation to occur. NADP, AcPyADP, SNADP, AADP, and oxidized glutathione all protected the enzyme from inactivation. 2'AMP, 3' AMP, 2'-phospho-5' AMP, 2'-phospho5'-ADP and 2'-phospho-ADP-ribose although all coenzyme-competitive inhibitors failed to protect the enzyme from N-ethylmaleimide inactivation. N-Phenyl and N-alkylmaleimides covalently modified two, of six available sulfhydryl groups per subunit. No other amino acid residues were modified. The reactivity of these sulfhydryl groups was at least two orders of magnitude higher than any reported for the N-ethylmaleimide reaction with many other 'essential sulfhydryl' enzymes. No change in the charge transfer band of the reduced enzyme was observed upon complete inactivation by N-ethyl, N-heptyl or N-phenylmaleimide. The retention of the charge transfer band after selective modification of two sulfhydryl groups suggests the involvement of a third reactive sulfhydryl group in the functioning of the yeast enzyme. No inactivation was observed when coenzymatically reduced enzyme was incubated with the site-specific sulfhydryl reagent, diazotized AADP.
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PMID:Simultaneous inactivation of the catalytic activities of yeast glutathione reductase by N-alkylmaleimides. 701 85

The histochemical creatine phosphokinase (CPK) tetrazolium test has been evaluated to detect recent human myocardial infarction in gross slices of the heart at necropsy. The demonstration of the lesion with this method has been assumed to result from local loss of CPK from the damaged myocardium. However, the present study indicates that the mechanism involved depends on localising NADPH tetrazolium reductase and not CPK. Phenazine methosulphate (PMS), when added to the incubating medium as an electron-acceptor to circumvent the tetrazolium-reductase (diaphorase) system, resulted in generalised false staining of the heart slice.
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PMID:Macroscopic enzyme histochemistry in myocardial infarction: artefactual nature of the creatine phosphokinase reaction. 707 68

The purification by affinity chromatography up to homogeneity and the properties of NAD-reductase from purple sulfur bacterium Thiocapsa roseopersicina, strain BBS, are described. The molecular weight of NAD-reductase is about 80000; pI is 3.9. The enzyme consists of two subunits. According to the stabilizing effect of FAD at preparative electrophoresis and the inhibitory effect of atebrine NAD-reductase is a flavoprotein. The bulk of the enzyme (about 75%) is localized in the cell periplasmic space. NAD-reductase is less thermostable and has a lower O2 stability as compared to the NADP-reductase from the same organism. The enzyme is specific to NADH ane catalyzes the menadione-reductase reaction, diaphorase reaction of benzyl viologen and methyl viologen reductions. In the presence of NADH NAD-reductase reduces cytochromes c552 and "c3" from T. roseopersicina and forms a complex with spinach ferredoxin.
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PMID:[Purification and properties of NAD-reductase from phototrophic bacterium Thiocapsa roseopersicina]. 723 99

The flavoprotein NADP+ reductase from spinach chloroplasts may form a ternary complex with one molecule of NADP+ and one molecule of ferredoxin. Spectroscopic titration studies show that the NADP+ binding site and the ferredoxin binding site are totally independent, that is previous binding of ferredoxin does not modify binding of NADP+, and conversely. Since NADP+ reductase conditions the diaphorase reaction, that is an electron transfer between NADPH and various acceptors such as ferricyanide, the binding of ferrocyanide and its possible interaction with NADP+ and ferredoxin has been studied. Ferrocyanide behaves as a competitive inhibitor with respect to both NADP+ and ferredoxin. This seems paradoxical since NADP+ and ferredoxin are independently bound at two different non-overlapping sites of the flavoprotein. This apparent paradox may be resolved by a theoretical analysis of the interactions between either ferrocyanide and NADP+, or ferrocyanide and ferredoxin. Theory shows that if ferrocyanide is non-specifically bound at two independent sites, namely the NADP+ and the ferredoxin binding sites, it appears competitive with respect to both NADP+ and ferredoxin, although ternary flavoprotein-ferredoxin-ferrocyanide and flavoprotein-NADP+-ferrocyanide complexes are formed. The binding constants of NADP+, ferredoxin and ferrocyanide for the enzyme have been determined. These results are discussed in connection with the possible mechanism of the diaphorase reaction.
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PMID:Complex-forming properties of spinach NADP+ reductase with ferredoxin, ferrocyanide and NADP+. 740 54

Bovine leukemia virus-transformed lamb embryo fibroblasts (line FLK) possess activity of DT-diaphorase of ca. 260 U/mg protein and similar levels of other NADP(H)-oxidizing enzymes: NADH:oxidase, 359 U/mg; NADPH:oxidase, 43 U/mg; NADH:cytochrome-c reductase, 141 U/mg; NADPH:cytochrome-c reductase, 43 U/mg. In general, the toxicity of aromatic nitrocompounds towards FLK cells increases on increase of single-electron reduction potentials (E1(1)) of nitrocompounds or the log of their reduction rate constants by single-electron-transferring enzymes, microsomal NADPH:cytochrome P-450 reductase (EC 1.6.2.4) and mitochondrial NADH:ubiquinone reductase (EC 1.6.99.3). No correlation between the toxicity and reduction rate of nitrocompounds by rat liver DT-diaphorase (EC 1.6.99.2) was observed. The toxicity is not significantly affected by dicumarol, an inhibitor of DT-diaphorase. Nitrocompounds examined were poor substrates for DT-diaphorase, being 10(4) times less active than menadione. Their poor reactivity is most probably determined by their preferential binding to a NADPH binding site, but not to menadione binding site of diaphorase. These data indicate that at comparable activities of DT-diaphorase and single-electron-transferring NAD(P)H dehydrogenases in the cell, the toxicity of nitrocompounds will be determined mainly by their single-electron reduction reactions.
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PMID:The toxicity of aromatic nitrocompounds to bovine leukemia virus-transformed fibroblasts: the role of single-electron reduction. 766 3

NADPH diaphorase activity was found in membrane of DMSO-induced differentiated human promyelocytic leukemia HL-60 cells. This membrane-bound diaphorase activity increased dramatically during differentiation of HL-60 cells. A dye reductase was extracted from membrane of DMSO-induced differentiated HL-60 cells with n-octyl glucoside and sodium cholate in the presence of several protease inhibitors such as PMSF, DIFP, TLCK, antipain, chymostatin, leupeptin, pepstatin A and trypsin inhibitor. The NADPH diaphorase was highly purified by two-stage sequential column chromatographies. The purified enzyme, showing both SOD-insensitive cytochrome c and NBT reductase activities, migrated with an apparent molecular mass of 77 kDa on SDS-PAGE. When the purification of this diaphorase was carried out in the presence of only three protease inhibitors, PMSF, DIFP and TLCK, a partially proteolyzed form of the diaphorase with a molecular mass of 68 kDa was prepared. The proteolyzed diaphorase exhibited only an NADPH-dependent cytochrome c reductase. The NADPH diaphorase gave a positive cross-reaction to polyclonal antibodies raised against microsomal NADPH-cytochrome P450 reductase from rabbit liver.
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PMID:Purification of an NADPH-dependent diaphorase from membrane of DMSO-induced differentiated human promyelocytic leukemia HL-60 cells. 769 24

We have cloned and sequenced the mouse NMO1 cDNA, which encodes the NAD(P)H:menadione oxidoreductase [also called NAD(P)H:(quinone acceptor) oxidoreductase; quinone reductase; azo dye reductase; DT diaphorase; EC 1.6.99.2]. The cDNA is 1528 bp in length excluding the poly(A+) tail, and has 5' and 3' nontranslated regions of 108 bp and 595 bp, respectively. The deduced protein contains 274 amino acids, including the first methionine (M(r) = 30,959). The mouse NMO1 protein is: 94% similar to the rat NMO1 and 86.5% to the human NMO1 proteins; 49.3% identical to the human NQO2 protein; and < 20% similar to several dozen other proteins in the quinone oxidoreductase superfamily. Southern hybridization analysis of mouse DNA reveals that the Nmo1 gene is likely to span less than a total of 20 kb. The Nmo1 gene is highly inducible by 2,3,7,8,-tetrachlorodibenzo-p-dioxin (dioxin; TCDD) in mouse liver and mouse cell cultures. TCDD inducibility of NMO1 is detectable at 12 and 18 days of gestation, but markedly elevated at 1-3 weeks post partum as compared with the 6- and 12-week-old mouse. NMO1 mRNA levels are strikingly elevated in the untreated mouse hepatoma Hepa-1c1c7 mutant line c37 lacking CYP1A1 (aryl hydrocarbon hydroxylase) activity, and in the untreated 14CoS/14CoS mouse cell line having an 'oxidative stress response' caused by homozygous deletion of about 3800 kb on chromosome 7. Previous work and the data in this report show that the murine Nmo1 gene is regulated by three distinct mechanisms: CYP1A1 metabolism-dependent repression, Ah receptor-mediated induction by TCDD, and activation by the chromosome 7-mediated oxidative stress response.
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PMID:Mouse dioxin-inducible NAD(P)H: menadione oxidoreductase: NMO1 cDNA sequence and genetic differences in mRNA levels. 770 40

A major and a minor ascorbate free radical (AFR) reductase were separated from the soluble fraction in the human lens cortex by DEAE-cellulose ion-exchange column chromatography. These AFR reductases also exhibited diaphorase activity using dichlorophenolindophenol and ferricyanide as electron acceptors. The major AFR reductase was partially purified by 5'AMP-Sepharose 4B affinity column chromatography. This partially purified AFR reductase showed a single band of diaphorase activity in native polyacrylamide disc gel electrophoresis. This activity band corresponded to the major protein observed in protein staining by Coomassie Brilliant Blue. However, the protein staining by Coomassie Brilliant Blue showed this activity band surrounded by diffused staining. Molecular weight of the partially purified AFR reductase was determined to be 32 kDa by gel filtration, and the apparent Km value for AFR was about 15 microM. This major lens AFR reductase could be distinguished from soluble Neurospora, Euglena and cucumber AFR reductases, and from two ubiquitous enzymes with reduction activity of AFR and/or foreign compounds, ie, NADH-cytochrome b5 reductase and DT-diaphorase, by their molecular weights, Km values and/or ion-exchange chromatographic behaviors.
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PMID:Soluble ascorbate free radical reductase in the human lens. 793 90

A dye reductase activity, independent of the production of superoxide, is induced in membranes prepared from stimulated human neutrophils or during activation of NADPH oxidase in a cell-free system. This diaphorase activity was greater under anaerobic as opposed to aerobic conditions. The activity has an absolute requirement for the membrane components of the oxidase, but does not appear to have an absolute dependence for the 47-kDa cytosolic factor p47-phox, suggesting the oxidase can be converted to a partial state of activation in the absence of this factor. The dye-reductase activity was inhibited at low concentration by the oxidase inhibitor, diphenylene iodonium. The electron acceptor, iodonitrotetrazolium violet (2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyltetrazolium chloride) is both a substrate and a mixed inhibitor of NADPH oxidation.
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PMID:The superoxide-generating system of human neutrophils possesses a novel diaphorase activity. Evidence for distinct regulation of electron flow within NADPH oxidase by p67-phox and p47-phox. 806 77


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