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:1.6.5.2 (NQO1)
6,196 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A simple three-step method was established for the purification of NAD(P)H dehydrogenase (quinone) ('DT-diaphorase', EC 1.6.99.2) from rat liver by affinity chromatography with a recovery of above 50%. The final enzyme preparation was purified about 750-fold and was electrophoretically homogeneous. Gel filtration showed that the enzyme had a mol.wt. of about 55 000, and one molecule of FAD was found per 55 000 mol.wt. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis gave a mol.wt. of about 27 000. Two N-terminal amino acids, asparagine/aspartic acid and glutamine/glutamic acid, were found in about equal yield, suggesting the presence of two non-identical polypeptide chains in the enzyme. NAD(P)H dehydrogenase was selectively removed by this affinity-chromatographic method from a microsomal carboxylation system. The system, which was solubilized by detergent and is dependent on vitamin K (2-methyl-3-phytyl-1,4-naphthaquinone or analogues with other side chains), lost its activity on the removal of the enzyme. The activity can be completely restored to the system by adding purified cytoplasmic NAD(P)H dehydrogenase or by using the quinol form of vitamin K1 (2-methyl-3-phytyl-1,4-naphthaquinol).
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PMID:NAD(P)H dehydrogenase and its role in the vitamin K (2-methyl-3-phytyl-1,4-naphthaquinone)-dependent carboxylation reaction. 62 56

Site-directed mutagenesis was utilized to identify binding sites for NAD(P)H and dicumarol in rat liver NAD(P)H:quinone oxidoreductase (NQOR, EC 1.6.99.2). The mutant cDNA clones were generated by a procedure based on the polymerase chain reaction and were expressed in Escherichia coli. The mutant enzymes were purified to apparent homogeneity as judged by SDS-polyacrylamide gel electrophoresis and were found to contain 2 FADs/enzyme molecule identical with that of the wild-type NQOR. Purified mutant enzymes Y128D, G150F, G150V, S151F, and Y155D showed dramatic decreases in activities in the reduction of dichlorophenolindophenol in comparison with the activities of the wild-type enzyme, whereas the activities of F124L, T127V, T127E, Y128V, Y128F, S151A, and Y155V were similar to those of NQOR. Enzyme kinetic analysis revealed that the Km values of T127E, Y128D, G150F, G150V, S151F, and Y155D were, respectively, 4-, 2-, 13-, 5-, 26-, and 19-fold higher than the Km of NQOR for NADPH, and were, respectively, 2-, 3-, 7-, 3-, 20-, and 11-fold higher than that of NQOR for NADH. The kcat values of Y128D, G150F, and G150V were also much lower than those of NQOR, but the kcat values of other mutants were similar to those of the wild-type enzyme. The Km values of the mutants for dichlorophenolindophenol were the same or slightly higher than that of NQOR. The apparent inhibition constants (Ki) for dicumarol on Y128V and F124L were elevated 12 and 8 times, respectively. Similar, but smaller, changes on Ki for 4-hydroxycoumarin were also observed. This study demonstrated that residues Gly150, Ser151, and Tyr155 in the glycine-rich region of NQOR are essential for NADPH and NADH binding and Tyr128 is important for dicumarol binding. Based on the results of the study, it is proposed that the glycine-rich region of the enzyme, along with other residues around the region, forms a beta sheet-turn-alpha helix structure important for the binding of the pyrophosphate group of NADPH and NADH.
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PMID:Identification of a glycine-rich sequence as an NAD(P)H-binding site and tyrosine 128 as a dicumarol-binding site in rat liver NAD(P)H:quinone oxidoreductase by site-directed mutagenesis. 138 97

Diaphorase-1 and diaphorase-2 were isolated from two Drosophila species, D. virilis and D. melanogaster, and purified by gel filtration, affinity chromatography, immunoaffinity chromatography, and ion-exchange chromatography. The molecular weights of both enzymes were the same in each species. The molecular weight of diaphorase-1 was the same under both denaturating and nondenaturating conditions, close to 60,000, indicating a monomeric structure. Sodium dodecyl sulfate (SDS) electrophoresis of the purified diaphorase-2 revealed the presence of a single protein band of 55,000 Da, while the molecular weight of the native enzyme was found to be 67,000. The two diaphorases were further characterized by their pH optima, isoelectric points, and kinetic parameters, and antibodies were raised in rabbits against the purified enzymes from D. virilis. The antibodies showed no cross-reactions but recognized the corresponding diaphorases in D. melanogaster and D. novamexicana as well as D. virilis. The data obtained confirmed the hypothesis of an independent genetic control of diaphorase-1 and diaphorase-2 in Drosophila.
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PMID:Purification and characterization of diaphorases from some Drosophila species. 161 84

A prokaryotic expression plasmid, pKK-DT2, containing the cDNA of rat liver NAD(P)H:quinone-acceptor oxidoreductase (EC 1.6.99.2; DT-diaphorase) was constructed and used to transform Escherichia coli strain JM109. The rat liver quinone reductase was expressed in strain in JM109 and was inducible with isopropyl beta-D-thiogalactopyranoside (IPTG). The expressed rat protein was purified by affinity chromatography and had kinetic and physical properties identical with the protein purified from rat liver in that it could utilize either NADH or NADPH as the electron donor and its activity was inhibited by dicoumarol. In addition, we have generated four mutants, Arg-177----His (R177H), Arg-177----Ala (R177A), Arg-177----Cys (R177C) and Arg-177----Leu (R177L), using this expression system. Several of the mutants behaved anomalously on SDS/PAGE, but all of the mutant proteins had the expected M(r) as determined by electrospray m.s. These results and those obtained from enzyme kinetic analysis, u.v./visible absorption spectral analysis, and flavin and tryptophan fluorescence analysis of the wild-type enzyme and four mutants indicated that mutations at Arg-177 changed the conformation of the enzyme, resulting in a decrease in enzyme activity. Replacing Arg-177 with leucine altered the protein conformation and decreased FAD incorporation.
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PMID:Expression of rat liver NAD(P)H:quinone-acceptor oxidoreductase in Escherichia coli and mutagenesis in vitro at Arg-177. 162 1

A novel RNA component with oxidoreductase activity (diaphorase activity) has been purified from an RNA fraction of Torula yeast. The RNA component was obtained in a 0.05% yield by a series of steps, SDS-phenol extraction, nuclease P1 digestion, alkaline phosphatase digestion, anion exchange chromatography, and HPLC on an ODS-column.
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PMID:Search for novel RNA catalysts. An RNA component with oxidoreductase activity. 210 15

It was found that when Escherichia coli is grown in the presence of 0.2-0.3 mM menadione (2-methyl-1,4-naphthoquinone), an FMN-dependent NADH-quinone reductase increases more than 20-fold in the cytoplasmic fraction. The menadione-induced quinone reductase was isolated from the cytoplasmic fraction of induced cells. The purified enzyme had an Mr of 24 kDa on SDS-polyacrylamide gel electrophoresis. The enzyme required flavin as a cofactor and a half-maximum activity was obtained with 0.54 microM FMN or 16.5 microM FAD. The enzyme had a broad pH optimum at pH 7.0-8.0 and reacted with NADH, but not with NADPH. The reaction followed a ping-pong mechanism and the intrinsic Km values for NADH and menadione were estimated to be 132 microM and 2.0 microM, respectively. Dicoumarol was a simple competitive inhibitor with respect to NADH with a Ki value of 0.22 microM. The electron acceptor specificity of this enzyme was very similar to that of NAD(P)H: (quinone acceptor) oxidoreductase (EC 1.6.99.2, DT-diaphorase) from rat liver. Since menadione is reduced by the two-electron reduction pathway to menadiol, the induction of this enzyme is likely to be an adaptive response of E. coli to partially alleviate the toxicity of menadione.
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PMID:Characterization of FMN-dependent NADH-quinone reductase induced by menadione in Escherichia coli. 211 86

Methylenetetrahydrofolate reductase from human cadaver liver was purified to homogeneity. The purified enzyme had a molecular mass of 150 kDa. On SDS-polyacrylamide gel electrophoresis it was dissociated into a single fragment with a molecular mass of 39 kDa. In contrast, fresh lymphocyte enzyme extract showed a major band with a molecular mass of 75 kDa and a minor band of 39 kDa. Fresh liver enzyme was inhibited by S-adenosylmethionine while the purified enzyme from human cadaver liver was not inhibited. These observations suggest that human methylenetetrahydrofolate reductase is composed of two identical subunits of 75 kDa each but is cleaved into a major single band due to autolysis in cadaver liver. The purified cadaver enzyme was a FAD-specific protein. The pH optimum was 6.6 for methylenetetrahydrofolate-NADPH oxidoreductase, 6.5 for methyltetrahydrofolate-menadione oxidoreductase, and 7.2 for NADP-menadione oxidoreductase. The Km values of human liver methylenetetrahydrofolate reductase were 17 microns for NADPH and 38 microns for methyltetrahydrofolate in the reduction of menadione, and 12 microns for NADPH in the reduction of methylenetetrahydrofolate.
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PMID:Purification and characterization of methylenetetrahydrofolate reductase from human cadaver liver. 238 27

The flavoprotein DT-diaphorase (EC 1.6.99.2) is believed to play an important role in the body's defense system. This enzyme has been purified 13,000-fold with a recovery of 58% from a cytosolic fraction of abdominal fat obtained from an obese patient undergoing elective surgery. Purification of the enzyme to electrophoretic homogeneity was achieved after two chromatographic steps: (1) affinity chromatography on azodicumarol Sepharose 6B; (2) anion exchange chromatography on DEAE Sephacel. The enzyme exhibits a monomer molecular mass of 32 kDa in SDS-PAGE and has 1 FAD prosthetic group per 32 kDa monomer. The FAD prosthetic group appears to be firmly attached to the apoproprotein. The enzyme reduces azodyes and quinones and demonstrates a broad substrate specificity. The enzyme has characteristics that are similar to DT-diaphorase purified from rodent liver, especially the rat liver enzyme. Estimated Km values for NADH, NADPH and menadione are 200, 140 and 3.3 microM, respectively. Vmax values for these substrates in the same order are 762, 667 and 294 mumol/mg.min. Dicumarol and warfarin exhibited competitive inhibition with pyridine nucleotides. The inhibition constants (Ki) for the drugs were estimated to be 10 nM and 2.2 microM, respectively. When compared to several other tissues, abdominal fat has one of the highest DT-diaphorase activities (Martin, L.F., Patrick, S.D. and Wallin, R. (1987) DT-diaphorase in morbidly obese patients. Cancer Lett., 36, 341-347), but the specific role of the enzyme in human fat is unknown.
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PMID:Human DT-diaphorase, a potential cancer protecting enzyme. Its purification from abdominal adipose tissue. 246 Feb 16

Walker tumour cells in vivo or in vitro are exceptionally sensitive to the monofunctional alkylating agent 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB 1954) (Cobb LM et al., Biochem Pharmacol 18: 1519-1527, 1969). CB 1954 forms DNA interstrand crosslinks in a time-dependent manner in Walker tumour cells but not in non-toxically affected Chinese hamster V79 cells [(Roberts JJ et al., Biochem Biophys Res Commun 140: 1073-1078, 1986)]. However, co-culturing Chinese hamster V79 cells with Walker cells in the presence of CB 1954 renders the hamster cells sensitive to CB 1954 and leads to the formation of interstrand crosslinks in their DNA, findings indicative of the formation by Walker cells of a diffusible toxic metabolite of CB 1954. A flavoprotein, of molecular weight 33.5 kDa as estimated by SDS-polyacrylamide gel electrophoresis, has been isolated from Walker cells and identified as a form of NAD(P)H dehydrogenase (quinone) (DT diaphorase, EC 1.6.99.2). This enzyme, in the presence of NADH or NADPH, catalyses the aerobic reduction of CB 1954 to 5-(aziridin-1-yl)-4-hydroxylamino-2-nitrobenzamide. This new compound can form interstrand crosslinks in the DNA of Chinese hamster V79 cells to which it is also highly toxic.
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PMID:A new cytotoxic, DNA interstrand crosslinking agent, 5-(aziridin-1-yl)-4-hydroxylamino-2-nitrobenzamide, is formed from 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB 1954) by a nitroreductase enzyme in Walker carcinoma cells. 320 2

The enzyme ferredoxin:NADP+ oxidoreductase (EC 1.18.1.2) from whole filaments of Anabaena cylindrica can be separated into four major fractions by chromatography on phosphocellulose; chromatography using ferredoxin-Sepharose 4B proved to be less satisfactory in separating the fractions. The purified fractions, designated 1, 2, 3 and 4, all showed diaphorase and ferredoxin-dependent cytochrome c reductase activity. The major fractions present were 2 and 3 which were each obtained in an electrophoretically homogeneous state (forms 2 and 3) and represented 30-37% and 30-42%, respectively, of the total enzyme activity. Each was a monomeric species with a molecular weight of approx. 33 000 as determined by gel filtration and sodium dodecyl (SDS)-polyacrylamide gel electrophoresis. Evidence for the presence of a 70 000 molecular weight dimer was also obtained. Forms 2 and 3 had isoelectric points of 5.75 and 6.0, respectively, had similar kinetic properties and were flavoproteins. Extracts of isolated heterocysts showed no form 2 or 3 activity but contained a single form which closely resembled one of the species present in fraction 4; fraction 1 may have been a purification artifact because it was not detected in crude extracts of the cyanobacterium.
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PMID:Molecular heterogeneity of ferredoxin:NADP+ oxidoreductase from the cyanobacterium Anabaena cylindrica. 678


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