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)

We have reported previously that enzymes present in the Sp 107 rat mammary carcinoma catalyse doxorubicin quinone reduction (QR) to 7-deoxyaglycone metabolites in vivo [Willmott and Cummings, Biochem Pharmacol 36: 521-526, 1987]. In order to provide insights into the role of QR in the antitumour mechanism of action of doxorubicin, we have attempted in this work to identify the enzyme(s) responsible. NAD(P)H: (quinone acceptor) oxidoreductase (DT-diaphorase) was the major quinone reductase in the tumour accounting for approximately 70% of all the activity measured in microsomes and cytosols (microsomal activity, 28.4 +/- 4.6 nmol/min/mg; cytosolic activity, 94.3 +/- 11.9 nmol/min/mg). Its presence was confirmed by western blot analysis. Low levels of NADH cytochrome b5 reductase (15.6 +/- 6.3 nmol/min/mg) and NADPH cytochrome P450 reductase (14.5 +/- 4.0 nmol/min/mg) were detectable in microsomes. The presence of the latter was confirmed by western blot analysis. Pretreatment of tumours with doxorubicin (48 hr) at a therapeutic dose decreased the level of activity of all the reductases studied by at least 2-fold (P < 0.01, Student's t-test). Doxorubicin was shown not to be a substrate for purified rat Walker 256 tumour DT-diaphorase with either NADH or NADPH as co-factor and utilizing up to 20,000 units of enzyme/incubation but was confirmed to be a substrate for purified rat liver cytochrome P450 reductase. 7-Deoxyaglycone metabolite formation by purified cytochrome P450 reductase had an absolute requirement for NADPH as co-factor, was inhibited by molecular oxygen and dicoumarol (IC50 approx. 50 microM), and modulated by specific reductase antiserum. Reductive deglycoslation of doxorubicin to 7-deoxyaglycones was localized to the microsomal fraction of the Sp 107 tumour, with negligible activity being found in cytosols (NADH, NADPH and hypoxanthine as co-factors) and mitochondria (NADH and NADPH). The tumour microsomal enzyme had an absolute co-factor requirement for NADPH, was inhibited by oxygen and dicoumarol, and modulated by cytochrome P450 reductase antiserum. These data indicate strongly that NADPH cytochrome P450 reductase is the principal enzyme responsible for catalysing doxorubicin QR in the Sp 107 tumour.
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PMID:The enzymology of doxorubicin quinone reduction in tumour tissue. 147 82

Xanthine dehydrogenase (XDH) from the unicellular green alga Chlamydomonas reinhardtii has been purified to electrophoretic homogeneity by a procedure which includes several conventional steps (gel filtration, anion exchange chromatography and preparative gel electrophoresis). The purified protein exhibited a specific activity of 5.7 units/mg protein (turnover number = 1.9 .10(3) min-1) and a remarkable instability at room temperature. Spectral properties were identical to those reported for other xanthine-oxidizing enzymes with absorption maxima in the 420-450 nm region and a shoulder at 556 nm characteristic of molybdoflavoproteins containing iron-sulfur centers. Chlamydomonas XDH was irreversibly inactivated upon incubation of enzyme with its physiological electron donors xanthine and hypoxanthine, in the absence of NAD+, its physiological electron acceptor. As deduced from spectral changes in the 400-500 nm region, xanthine addition provoked enzyme reduction which was followed by inactivation. This irreversible inactivation also took place either under anaerobic conditions or whenever oxygen or any of its derivatives were excluded. Adenine, 8-azaxanthine and acetaldehyde which could act as reducing substrates of XDH were also able to inactivate it upon incubation. The same inactivating effect was observed with NADH and NADPH, electron donors for the diaphorase activity associated with xanthine dehydrogenase. In addition, partial activities of XDH were differently affected by xanthine incubation. We conclude that xanthine dehydrogenase inactivation by substrate is due to an irreversible process affecting mainly molybdenum center and that sequential and uninterrupted electron flow from xanthine to NAD+ is essential to maintain the enzyme in its active form.
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PMID:Purification and substrate inactivation of xanthine dehydrogenase from Chlamydomonas reinhardtii. 152 76

An FMN-dependent NADH-quinone reductase is induced in Escherichia coli by growing the cells in the presence of menadione (2-methyl-1,4-naphthoquinone). Since the properties of induced enzyme are very similar to those of NAD(P)H: (quinone-acceptor) oxidoreductase (EC 1.6.99.2), known as DT-diaphorase, from animal cells, structural requirements of quinone derivatives as an inducer of NADH-quinone reductase in E. coli were examined. Among quinone derivatives examined, it was found that 2-alkyl-1,4-quinone structure with C-3 unsubstituted or substituted with Br is critical as a common inductive signal. Michael reaction acceptors which have been reported to be strong inducers of DT-diaphorase in mouse hepatoma cells were not always effective inducers in E. coli. However, several compounds, such as 2-methylene-4-butyrolactone, methylacrylate and methyl vinyl ketone, showed a slight inductive activity. The efficient inducers of NADH-quinone reductase in E. coli contain 1,4-quinone structure as a part of the inductive signal. These compounds belong to Michael acceptors and are likely to conjugate with thiol compounds such as glutathione.
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PMID:Chemical structures critical for the induction of FMN-dependent NADH-quinone reductase in Escherichia coli. 154 1

Dietary composition is a major determinant of cancer risk in humans and experimental animals. Major and minor components of the diet may enhance or suppress the development of malignancy. Many dietary constituents also modify the metabolism of carcinogens by induction of enzymes involved in xenobiotic metabolism, and this is one well-established mechanism for modulating the risk of cancer. We have developed a simple system for rapid detection and measurement of the induction of enzymes that detoxify carcinogens (phase II enzymes), based on the direct assay of the activity of quinone reductase [NAD(P)H:(quinone-acceptor) oxidoreductase, EC 1.6.99.2] in murine hepatoma cells grown in microtiter plate wells. Survey of extracts of a variety of commonly consumed, organically grown vegetables for quinone reductase inducer activity identified crucifers (and particularly those of the genus Brassica) as singularly rich sources. It is therefore of interest that high consumption of these types of vegetables has been correlated with decreased cancer risk in humans. The assay system also measures toxicity, which was unrelated to inducer potency among the vegetable extracts examined. By use of mutant hepatoma cells (defective in regulation of certain cytochrome P-450 enzymes) selective (monofunctional) inducers of protective phase II enzymes can be distinguished from (bifunctional) inducers that also elevate cytochromes P-450 (phase I enzymes) and thereby pose the risk of carcinogen activation. The assay system therefore permits not only rapid detection of inducers of anticarcinogenic enzymes in the human diet but also elucidation of effects of storage and processing on inducer activities.
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PMID:Rapid detection of inducers of enzymes that protect against carcinogens. 154 2

Consumption of vegetables, especially crucifers, reduces the risk of developing cancer. Although the mechanisms of this protection are unclear, feeding of vegetables induces enzymes of xenobiotic metabolism and thereby accelerates the metabolic disposal of xenobiotics. Induction of phase II detoxication enzymes, such as quinone reductase [NAD(P)H:(quinone-acceptor) oxidoreductase, EC 1.6.99.2] and glutathione S-transferases (EC 2.5.1.18) in rodent tissues affords protection against carcinogens and other toxic electrophiles. To determine whether enzyme induction is responsible for the protective properties of vegetables in humans requires isolation of enzyme inducers from these sources. By monitoring quinone reductase induction in cultured murine hepatoma cells as the biological assay, we have isolated and identified (-)-1-isothiocyanato-(4R)-(methylsulfinyl)butane [CH3-SO-(CH2)4-NCS, sulforaphane] as a major and very potent phase II enzyme inducer in SAGA broccoli (Brassica oleracea italica). Sulforaphane is a monofunctional inducer, like other anticarcinogenic isothiocyanates, and induces phase II enzymes selectively without the induction of aryl hydrocarbon receptor-dependent cytochromes P-450 (phase I enzymes). To elucidate the structural features responsible for the high inducer potency of sulforaphane, we synthesized racemic sulforaphane and analogues differing in the oxidation state of sulfur and the number of methylene groups: CH3-SOm-(CH2)n-NCS, where m = 0, 1, or 2 and n = 3, 4, or 5, and measured their inducer potencies in murine hepatoma cells. Sulforaphane is the most potent inducer, and the presence of oxygen on sulfur enhances potency. Sulforaphane and its sulfide and sulfone analogues induced both quinone reductase and glutathione transferase activities in several mouse tissues. The induction of detoxication enzymes by sulforaphane may be a significant component of the anticarcinogenic action of broccoli.
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PMID:A major inducer of anticarcinogenic protective enzymes from broccoli: isolation and elucidation of structure. 154 3

Resting suspensions of cells of Saccharomyces cerevisiae grown in iron-rich or iron-deficient conditions were studied by following the fluorescence emission changes (lambda em. 400-460 nm, lambda exc. 300-340 nm) occurring in these suspensions upon addition of glucose and ferric iron. The results show that, in addition to NAD(P)H, metabolites of the aromatic amino acid pathway interfere with the fluorescence measurements, and that they could be involved in ferric iron reduction. Wild-type strains of S. cerevisiae are known to excreted anthranilic acid and 3-hydroxyanthranilic acid in response to glucose. The major fluorescing compound excreted by a chorismate-mutase-deficient mutant strain of S. cerevisiae was identified as anthranilic acid. The excretion of anthranilic and 3-hydroxyanthranilic acids was correlated with the ferric-reducing capacity of the extracellular medium. Excretion during growth was much greater by cells cultured in iron-rich medium than by cells grown in iron-deficient medium. The possibility was examined that a link could exist between the biosynthesis of aromatics and the ferri-reductase activity of the cells, via chorismate synthase and its putative diaphorase-associated activity. Two ferri-reductase-deficient mutants excreted much less 3-hydroxyanthranilate than did the parental wild-type strains. However, the ferri-reductase activity of a chorismate-synthase-deficient mutant was comparable to that of the parental strain.
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PMID:Excretion of anthranilate and 3-hydroxyanthranilate by Saccharomyces cerevisiae: relationship to iron metabolism. 155 59

The effects of three acid condensation products of indole-3-carbinol (I3C), i.e. 3,3'-diindolylmethane (DIM), 5,6,11,12,17,18-hexahydrocyclonona[1,2-b:4,5-b':7,8-b"]tri-indole (CTI) and 2,3-bis[3-indolylmethyl]indole (BII), on cytochrome P450 and phase II enzymes were studied in primary cultures of rat and cynomolgus monkey liver cells. In rat hepatocytes all three indole derivatives dose-relatedly induced the ethoxyresorufin O-dealkylation (EROD) activity (to 24-fold) and 7 alpha-hydroxylation of testosterone (to 4-fold), whereas all three decreased the 16 alpha- and 2 alpha-testosterone hydroxylation (DIM to 60%, CTI and BII to a mere 5% of the control cells). Treatment of monkey hepatocytes with DIM and BII enhanced the EROD activity to 6- and 9-fold, respectively. Furthermore, BII decreased the 6 beta-hydroxylation of testosterone (to 60% of the untreated cultures) in monkey cells. Phase II enzymes were also affected. In rat hepatocytes DIM, CTI and BII enhanced DT-diaphorase (DTD) (= NAD(P)H-quinone reductase) activity, and DIM and BII the glucuronidation of 1-naphthol. In monkey cells BII only enhanced DTD, and no changes were observed in the glucuronidation of 1-naphthol after treatment with either DIM or BII. The indole derivatives did not affect glutathione S-transferase activity and sulfation of 1-naphthol in either rat or monkey hepatocytes. These results identify two novel acid condensation products of I3C, CTI and BII, as potent compounds in affecting biotransformation in rat as well as in monkey hepatocytes.
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PMID:Acid reaction products of indole-3-carbinol and their effects on cytochrome P450 and phase II enzymes in rat and monkey hepatocytes. 156 68

1. Dihydrodiol dehydrogenase activities were investigated in rabbit liver. Using a five-step purification scheme, eight isoenzymes of dihydrodiol dehydrogenase with isoelectric points of 5.55-9.3 and promoter molecular masses of 34-35 kDa were purified to apparent homogeneity and designated CF-1 to CF-6, CM-1 and CM-2. 2. CF-1 and CF-2 had near-neutral isoelectric points of 7.4 and 6.8 and molecular masses of about 125 kDa in the native state. Both enzymes readily accepted NAD+ as well as NADP+ as coenzymes, had relatively low Km values of 0.33 mM and 0.47 mM for benzene dihydrodiol and resembled previously described carbonyl reductases in their substrate specificity towards ketones and quinones. 3. CF-5 and CF-6 had acidic isoelectric points of 5.9 and 5.55 and native molecular masses of approximately 60 kDa. They displayed a strong preference for NADP(H) as coenzyme and had high Km and Vmax with benzene dihydrodiol. Since these enzymes reduced p-nitrobenzaldehyde and glucuronic acid efficiently, they appeared to be closely related to aldehyde reductase. 4. CF-4 had a high 3 alpha-hydroxysteroid dehydrogenase activity for the diagnostic substrate androsterone, a moderate activity for other 3 alpha-hydroxysteroids as well as 17 alpha-hydroxysteroids, and relatively low activities for 3 beta-hydroxysteroids and 17 beta-hydroxysteroids. CF-5 and CM-1 had high 17 beta-hydroxysteroid dehydrogenase activity for the diagnostic substrate 5 alpha-dihydrotestosterone, and low to moderate activities for other 17 beta-hydroxysteroids as well as 3 alpha-hydroxysteroids. 5. The isoenzyme CM-2 had an isoelectric point of 9.3 and was a very active quinone reductase with phenanthrene-9,10-quinone as substrate. It was potently inhibited by phenobarbital. 6. We conclude that the dihydrodiol dehydrogenase activities of rabbit liver are associated with aldehyde and carbonyl reductase and with 3 alpha-hydroxysteroid and 17 beta-hydroxysteroid dehydrogenases.
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PMID:Dihydrodiol dehydrogenase activities of rabbit liver are associated with hydroxysteroid dehydrogenases and aldo-keto reductases. 157 98

Rats fed an ethanol-containing diet for 4 weeks showed a 3- to 5-fold increase over isocalorically pair-fed controls with respect to cytosolic NAD(P)H-quinone oxidoreductase (NQOR) (E.C.1.6.99.2) with both menadione and dichlorophenol-indophenol as substrates. Rates of NAD(P)H-dependent p-nitrosophenol (pNSP) reduction catalyzed by rat liver cytosolic fractions were increased 1.5- to 2-fold upon pretreatment of the animal with ethanol. NQOR contributed almost exclusively to the NADPH-dependent C-nitrosoreductase activity in cytosol as judged by the strong inhibition of the reaction by dicoumarol. In contrast, NADH-dependent C-nitrosoreductase activity was inhibited 70-80% by pyrazole and thus may be attributed mainly to alcohol dehydrogenase(s). Highly purified rat liver cytosolic NQOR catalyzed the NADH- and NADPH-dependent reduction of pNSP to p-aminophenol. We therefore suggest that ethanol ingestion enhances the reduction of the C-nitrosoaromatics formed upon cytosolic metabolism of arylamines or nitroarenes by two mechanisms. Increased NADPH-dependent reduction is mediated by the induction of cytosolic NQOR while an NADH-dependent pathway responds to the increased availability of reduced cofactor upon ethanol ingestion and involves mainly the alcohol dehydrogenase-mediated reduction of such compounds.
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PMID:Role of cytosolic NAD(P)H-quinone oxidoreductase and alcohol dehydrogenase in the reduction of p-nitrosophenol following chronic ethanol ingestion. 158 50

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


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