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)

Ubiquinone (UQ) reductase activity which reduces UQ to ubiquinol (UQH2) in rat tissues was roughly proportional to the UQH2/total UQ ratio in respective tissues. The highest activity was found in the liver, showing the highest UQH2/total UQ ratio. A greater part of liver UQ reductase activity was located in the cytosol. Within a week, the liver UQ reductase activity decreased by 80% even at -20 degrees C. The DT-diaphorase activity was stable. UQ reductase required NADPH as the hydrogen donor and was not inhibited by a less than 1 microM concentration of dicoumarol. There was no stimulation of UQ reductase in the presence of bovine serum albumin nor in Triton X-100. Yet, both stimulated DT-diaphorase. As a result, UQ reductase appeared to be a novel NADPH-UQ oxidoreductase and responsible for the UQ redox state in liver.
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PMID:A novel ubiquinone reductase activity in rat cytosol. 146 65

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

A nitroreductase enzyme has been isolated from Escherichia coli B. This enzyme is an FMN-containing flavoprotein with a molecular mass of 24 kDa and requires either NADH or NADPH as a cofactor. Partial protein sequence analysis showed extensive homology with the "classical nitroreductase" of Salmonella typhimurium and a nitroreductase induced in Enterobacter cloacae. In common with the Salmonella enzyme, the E. coli B enzyme is capable of reducing nitrofurazone. The E. coli nitroreductase is also capable of reducing the anti-tumour agent CB1954 [5-(aziridin-1-yl)-2,4-dinitrobenzamide], a property shared with the mammalian enzyme DT diaphorase [NAD(P)H dehydrogenase (quinone)] as isolated from Walker cells. The reduction of CB1954 by the E. coli enzyme results in the generation of cytotoxic species. Both enzymes also share the properties of being able to reduce quinones and are both inhibited by dicoumarol. The nitroreductase is a more active enzyme against CB1954 (kcat = 360 min-1) than Walker DT diaphorase (kcat = 4 min-1) and also has a lower Km for NADH (6 vs 75 microM).
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PMID:The bioactivation of 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB1954)--I. Purification and properties of a nitroreductase enzyme from Escherichia coli--a potential enzyme for antibody-directed enzyme prodrug therapy (ADEPT). 147 94

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

We previously reported the expression of a full-length cDNA complementary to a rat liver NAD(P)H:quinone oxidoreductase (EC 1.6.99.2) mRNA in Escherichia coli (Q. Ma, R. Wang, C. S. Yang, and A. Y. H. Lu, 1990, Arch. Biochem. Biophys. 283, 311-317). Since cysteine residues have been suggested to be important for the catalysis of flavoproteins and a lysine residue at position 76 in NAD(P)H:quinone oxidoreductase has been proposed to be involved in electron transfer of the enzyme, we investigated the roles of lysine 76 and cysteine 179 of this enzyme in catalysis by site-directed mutagenesis. Mutant cDNA clones replacing lysine 76 with valine (K76V) and cysteine 179 with alanine (C179A) were generated by a procedure based on the polymerase chain reaction. The mutant enzymes were expressed in E. coli. The cytosolic activities of the K76V and C179A mutants were 50 and 25% of that of the wild type (DTD), due to lower levels of the mutant proteins as shown by immunoblot analysis. The mutant proteins were purified to apparent homogeneity. The purified K76V and C179A mutant enzymes maintained full activities of 2,6-dichlorophenolindophenol (DCIP) reduction compared with that of the wild type. The mutant enzymes exhibited kinetic parameters for DCIP, NADH, and NADPH similar to those of DTD except that, with K76V, the Km for NADPH was doubled. Both mutant proteins contained two molecules of FAD per enzyme molecule. Dicumarol inhibited K76V and C179A mutant activities to greater than 90% at a concentration of 10(-7) M. Heat stability studies showed that C179A was much more sensitive to inactivation at 37 degrees C than both the wild-type and K76V enzymes. It is concluded from this study that lysine 76 and cysteine 179 are not essential in catalysis and in the binding of FAD, DCIP, and dicumarol. However, lysine residue 76 appears to play a role in NADPH binding and cysteine residue 179 is important in maintaining the stability of the enzyme.
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PMID:Site-directed mutagenesis of rat liver NAD(P)H: quinone oxidoreductase: roles of lysine 76 and cysteine 179. 156 99

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

The topographic distribution of nicotinamide adenine dinucleotide-diaphorase (NADPH-d) stained profiles in the amygdala of the human and new world monkey (Saimiri sciureus) were studied histochemically. Fiber and terminal staining were heterogeneously distributed within the amygdala. The most intense staining occurred in the basolateral subdivision, consisting of the lateral, basolateral and accessory basal nuclei. Moderate staining intensity was observed throughout the cortical and media nuclei and cortical transition area, constituents of the corticomedial subdivision. The central amygdaloid area was characterized by minimal NADPH-d histochemical reactivity. NADPH-d positive neurons were pleomorphic and divisible into two classes based on their staining characteristics: intensely or lightly stained neurons. Their distribution was generally complementary, with the majority of intensely stained neurons occupying the basolateral subdivision. There were no appreciable species differences in the patterns of neuronal, fiber and terminal staining between monkey or human amygdala. These results may be relevant to our understanding of the selective vulnerability of neural systems within the human amygdala in neurodegenerative diseases.
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PMID:Reduced nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) profiles in the amygdala of human and New World monkey (Saimiri sciureus). 160 98

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

Human or rat microsomal 5 alpha-reductase activity, as measured by enzymic conversion of testosterone into 5 alpha-dihydrotestosterone or by binding of a competitive inhibitor, [3H]17 beta-NN-diethulcarbamoyl-4-methyl-4-aza-5 alpha-androstan-3-one ([3H]4-MA) to the reductase, is inhibited by low concentrations (less than 10 microM) of certain polyunsaturated fatty acids. The relative inhibitory potencies of unsaturated fatty acids are, in decreasing order: gamma-linolenic acid greater than cis-4,7,10,13,16,19-docosahexaenoic acid = cis-6,9,12,15-octatetraenoic acid = arachidonic acid = alpha-linolenic acid greater than linoleic acid greater than palmitoleic acid greater than oleic acid greater than myristoleic acid. Other unsaturated fatty acids such as undecylenic acid, erucic acid and nervonic acid, are inactive. The methyl esters and alcohol analogues of these compounds, glycerols, phospholipids, saturated fatty acids, retinoids and carotenes were inactive even at 0.2 mM. The results of the binding assay and the enzymic assay correlated well except for elaidic acid and linolelaidic acid, the trans isomers of oleic acid and linoleic acid respectively, which were much less active than their cis isomers in the binding assay but were as potent in the enzymic assay. gamma-Linolenic acid had no effect on the activities of two other rat liver microsomal enzymes: NADH:menadione reductase and glucuronosyl transferase. gamma-Linolenic acid, the most potent inhibitor tested, decreased the Vmax. and increased Km values of substrates, NADPH and testosterone, and promoted dissociation of [3H]4-MA from the microsomal reductase. gamma-Linolenic acid, but not the corresponding saturated fatty acid (stearic acid), inhibited the 5 alpha-reductase activity, but not the 17 beta-dehydrogenase activity, of human prostate cancer cells in culture. These results suggest that unsaturated fatty acids may play an important role in regulating androgen action in target cells.
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PMID:Inhibition of steroid 5 alpha-reductase by specific aliphatic unsaturated fatty acids. 163 46

A highly active preparation of rat liver dihydrodiol/3 alpha-hydroxysteroid dehydrogenase was obtained using a newly developed, rapid purification scheme involving affinity chromatography on Red Sepharose. Depending on the coenzyme present, the purified enzyme was found to catalyse the oxidation of dihydrodiols and steroids or the reduction of substrates with carbonyl or quinone moieties. Using a wide range of synthetic quinones derived from polycyclic aromatic hydrocarbons (PAHs), we observed a pronounced regioselectivity of the quinone reductase activity. Good substrates were the o-quinones of phenanthrene, benz(a)anthracene, chrysene and benzo(a)pyrene with the quinonoid moiety in the K-region which were reduced at rates of 1-10 mumol/min.mg enzyme. 1,4-Benzoquinone, naphthalene-1,2-quinone and benz(a)anthracene-8,9-quinone were also reduced at high rates. In contrast, alkyl-substituted quinones such as duroquinone and menadione were poor substrates for the enzyme. During the enzymatic reduction of several o-quinones, but not 1,4-benzoquinone, we observed the oxidation of large amounts of NADPH and the consumption of molecular oxygen which is indicative of a redox-cycling process. Thus, the reduction of quinones of PAHs may lead to a facilitated conjugation and excretion of these highly lipophilic compounds, but may also initiate toxic processes due to the formation of reactive oxygen species.
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PMID:Quinone reduction and redox cycling catalysed by purified rat liver dihydrodiol/3 alpha-hydroxysteroid dehydrogenase. 164 48


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