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

Dihydrodiol dehydrogenase (DD; EC 1.3.1.20) will oxidize non-K-region trans-dihydrodiols of polycyclic aromatic hydrocarbons (PAHs), a reaction that can suppress the formation of PAHs) anti-diol epoxides or ultimate carcinogens. Using benzenedihydrodiol [(+/-)-trans-1,2-dihydroxy-3,5-cyclohexadiene] as a model substrate for trans-dihydrodiol metabolites of PAHs, 23 human liver and eight human lung samples were examined for enzyme activity. In human liver, enzyme activity could be measured spectrophotometrically and specific activities ranged from 0.16 to 6.1 nmol benzenedihydrodiol oxidized min/mg protein. Western blot analysis of human liver cytosol using rabbit anti-rat DD serum detected two bands of mol. wts 34,000 and 27,000. The former mol. wt is identical to that observed for the homogeneous rat liver enzyme. Gel-filtration experiments indicate that human liver DD activity elutes as a single peak and co-elutes with the purified rat liver enzyme, suggesting that the lower mol. wt species may be an artefact of degradation. Preparations of the human liver enzyme required NADP- for activity and were in general, insensitive to inhibition by dicoumarol, indomethacin and 6-medroxyprogesterone acetate. These properties distinguish the enzyme from alcohol dehydrogenase, quinone reductase and rat liver DD. In human lung, DD activity was barely detectable using a sensitive radiochemical assay in which the oxidation of benzenedihydrodiol to catechol is linked to catechol-O-methyl transferase using [3H]S-adenosyl methionine as methyl donor. Specific activities were approximately 1000th of that observed for human liver and ranged from 1 to 4 pmol benzenedihydrodiol oxidized/min/mg protein. Western blot analysis of lung cytosol detected three bands of mol. wts 34,000, 31,000 and 28,000. The relatively high levels of DD in human liver suggest that this enzyme may play an important role in PAH detoxication in this organ, while the low levels of DD in lung may contribute to the susceptibility of this tissue to PAH-induced carcinogenesis.
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PMID:Characterization of dihydrodiol dehydrogenase in human liver and lung. 219 14

The respiratory chain-linked NADH-quinone reductase (NQR) and NADH-ferricyanide dehydrogenase (NFD) were extracted from membranes of Escherichia coli by n-dodecyl octaethyleneglycol monoether detergent and purified by DEAE-Sephacel, DEAE-5PW and Bio-Gel HTP column chromatography. The purified NQR contained FAD as a cofactor, catalyzed the reduction of ubiquinone-1 (Q1) and reacted with NADH, but not with deamino-NADH (d-NADH), with an apparent Km of 48 microM. On the other hand, the purified NFD contained FMN as a cofactor, reacted with both NADH and d-NADH, and catalyzed the reduction of ferricyanide but not Q1. NFD showed a high affinity for both NADH and d-NADH with a Km of 7-9 microM. NFD was inactivated, whereas NQR was rather activated, by preincubation with an electron donor in the absence of electron acceptor. These properties were compared with those of activities observed with inverted membrane vesicles with special reference to the generation of inside-positive membrane potential (delta psi). It was found that d-NADH-reactive FMN-containing NFD is a dehydrogenase part of energy-generating NADH-quinone reductase complex. The FAD-containing NQR was very similar to that purified by Jaworowski et al. (Biochemistry (1981) 20, 2041-2047), and reduced Q1 without generating delta psi.
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PMID:Purification of NADH-ferricyanide dehydrogenase and NADH-quinone reductase from Escherichia coli membranes and their roles in the respiratory chain. 267 83

DT-Diaphorase purified from the liver cytosol of rats treated with a highly toxic PCB congener, 3,4,5,3',4'-pentachlorobiphenyl (PenCB), was compared to those from 3-methylcholanthrene (MC)-treated and untreated rats. Treatments with PenCB and MC resulted in about 8- and 7-fold increases of cytosolic DT-diaphorase activity, respectively. Purification of the enzyme preparations from untreated, and PenCB- and MC-treated rats were conducted by using DE-52, DEAE-Sephadex A-50, hydroxylapatite, and Bio-Gel P-150 column chromatographies. Both Sephadex G-100 gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that all of the final preparations from the three origins were homogeneous and had the same molecular weight of 59,000, consisting of two subunits with molecular weights of 30,000. Further studies on amino acid composition, Km value, optimum pH, and catalytic activities for various substrates also indicated that both PenCB- and MC-inducible DT-diaphorases were identical with that from the untreated rats. All three DT-diaphorases contained about 2 mol of FAD per mol of enzyme. Partial digestion of the enzymes by trypsin and subsequent analysis by HPLC revealed that the three preparations were indistinguishable. The identity among the three purified DT-diaphorases was finally confirmed by Ouchterlony immunodiffusion employing anti-serum raised against each enzyme preparation.
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PMID:Comparison of DT-diaphorases purified from the liver cytosol of untreated, and 3,4,5,3',4'-pentachlorobiphenyl- and 3-methylcholanthrene-treated rats. 312 17

The ferredoxin-NADP+ oxidoreductase of spinach chloroplasts was purified from a Triton X-100 thylakoid extract closely associated with an intrinsic polypeptide of 17.5 kDa. The 17.5-kDa polypeptide-reductase complex differs from soluble ferredoxin-NADP+ reductase in (a) its elution profile in an Affi-Gel blue column; (b) its behavior in isoelectric focusing electrophoresis; and (c) giving different immunoelectrophoretic arcs. The diaphorase activity of the purified complex showed the same pH profile of thylakoid-bound reductase. The curve changed to a form similar to that of soluble reductase after dissociation of the complex. Dissociation allowed separation of the components and was reversible. It is suggested that the 17.5-kDa intrinsic polypeptide is the reductase-binding protein and that it may play an important role in the physiological regulation of the reductase and of photosynthetic electron transport.
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PMID:Evidence for the existence of a thylakoid intrinsic protein that binds ferredoxin-NADP+ oxidoreductase. 673 31

Rat liver homogenates reduced ubiquinone (UQ)-10 to ubiquinol (UQH2)-10 in the presence of NADPH rather than NADH. This NADPH-dependent UQ reductase (NADPH-UQ reductase) activity that was not inhibited by antimycin A and rotenone, was located mainly in the cytosol fraction and its activity accounted for 68% of that of the homogenates. Furthermore, the NADPH-UQ reductase from rat liver cytosol efficiently reduced both UQ-10 incorporated into egg yolk lecithin liposomes, and native UQ-9 residing in rat microsomes, to the respective UQH2 form in the presence of NADPH. The gross redox ratios of UQH2-9/(UQ-9 + UQH2-9) in individual tissues of rat correlated positively with the log of their respective cytosolic NADPH-UQ reductase activities, while the redox ratios in every intracellular fraction from liver were at about the same level, irrespective of NADPH-UQ reductase activities in the respective fractions. The combined addition of rat liver cytosol and NADPH inhibited to a great extent 2,2'-azobis(2,4-dimethyl-valeronitrile)-induced lipid peroxidation of UQ-10-fortified lecithin liposomes and completely inhibited such peroxidation in the liposomes in which UQH2-10 replaced UQ-10. The NADPH-UQ reductase activity was clearly separated from DT-diaphorase (EC 1.6.99.2) activity by means of Cibacron Blue-immobilized Bio-Gel A-5m chromatography. In conclusion, the NADPH-UQ reductase in cytosol, which is a novel enzyme to our knowledge, was presumed to be responsible for maintaining the steady-state redox levels of intracellular UQ and thereby to act as an endogenous antioxidant in protecting intracellular membranes from lipid peroxidation that is inevitably induced in aerobic metabolism.
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PMID:Reduction of ubiquinone in membrane lipids by rat liver cytosol and its involvement in the cellular defence system against lipid peroxidation. 763 6

To study the regulation of the human detoxicating enzyme DT diaphorase (DTD) under hypoxic conditions, we examined the effects of heat and hypoxia, and their interaction, on the steady-state levels of mRNA and DTD enzyme activity in human colon adenocarcinoma HT29 cells. We found that a 1-h heat treatment (42.5 degrees C) markedly increased the specific activity of DTD. Elevated enzyme activity was observed within 30 min, peaked at 6 h, and had almost returned to baseline by 36 h. The effect of hypoxia alone on DTD enzyme activity developed more slowly, with a maximal response at 24 h, and return to baseline at 72 h. The effect of a 1-h heat treatment was not inhibited by subsequent hypoxic exposure for 8 h. The effect of hypoxia was also not inhibited by heat in any schedule. However, a 1-h exposure to heat during 8 h hypoxic exposure induced the transcriptional effects of heat treatment much earlier. Heat shock followed by hypoxic stress resulted in prolonged elevation of DTD activity similar to that observed with hypoxia alone. We found that DTD mRNA content was elevated with a time course concordant with that of the enzyme activity. These data suggest that hypoxia and heat shock induce expression of the DTD gene independently. The mechanism of heat effect on DTD gene expression was investigated. Gel retardation assays demonstrated the induction of a binding activity of heat-induced transcription factor(s) to heat shock elements following both heat and hypoxia.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Interaction of heat and hypoxia in modulating transcription of DT diaphorase in human colon adenocarcinoma cells. 818 Jan 25

To assess the potential differential lung tumour expression of NAD(P)H:quinone reductase (NQO1), the human (h) NQO1 promoter was characterized in gene transfer studies. A deletion panel of 5' flanking hNQO1 promoter constructs was made and tested in transient transfection assays in NSCLC and SCLC cell lines. The largest hNQO1 construct (-1539/+115) containing the antioxidant response element (ARE), exhibited robust levels of reporter activity in the NSCLC (H460, H520, and A549) cell lines and expression was over 12 to 77-fold higher than the minimal (-259/+115) promoter construct. In contrast, there was little difference in promoter activity between the largest and minimal promoter construct in the SCLC (H146, H82 and H187) cell lines. Deletion of the sites for NFkappaB and AP-2 and the XRE did not significantly affect hNQO1 promoter activity in either the NSCLC or SCLC cell lines. Robust promoter activity in NSCLC lines was mediated by a 359 bp segment of the proximal promoter that contained a canonical AP-1 binding site, TGACTCAG, within the ARE. Gel supershift assays with various specific Fos/Jun antibodies identified Fra1, Fra2 and Jun B binding activity in NSCLC cells to a promoter fragment (-477 to -438) spanning the AP-1 site, whereas SCLC do not appear to express functional Fra or Jun B. These results suggest a possible role for AP-1 activity in the differential expression of hNQO1 in NSCLC.
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PMID:DT-diaphorase activity in NSCLC and SCLC cell lines: a role for fos/jun regulation. 1020 77

beta-Naphthoflavone (beta-NF) is a widely used inducer of phase-I and phase-II enzymes controlled by aryl hydrocarbon receptor (AhR). Studies of competitive binding with (3)H-labelled 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD), 3-methylcholanthrene (3-MC) and benzo[a]pyrene (B[a]P) have shown that beta-NF is a high-affinity ligand for AhR and also for polycyclic aromatic hydrocarbon (PAH)-binding protein, both soluble proteins of rat liver in 8 S and 4 S fractions, respectively, of sucrose gradients. This study examined binding of [(3)H]beta-NF to liver cytosolic proteins of female Sprague-Dawley rats. Treatment of rats with beta-NF, 3-MC, TCDD or alpha-naphthoflavone (alpha-NF) increased the specific [(3)H]beta-NF binding to liver cytosol up to 125-fold that of vehicle (corn oil)-treated rats (<100 fmol/mg of protein). Sucrose gradients revealed a large 4 S and a small 8 S peak of radioactivity from [(3)H]beta-NF binding to cytosols of beta-NF-, 3-MC-, TCDD- or alpha-NF-treated rats. Whereas co-incubation with the unlabelled beta-NF eliminated both peaks, co-incubation with 2,3, 7,8-tetrachlorodibenzofuran (TCDF) eliminated only the 8 S peak. The sucrose density gradient from [(3)H]TCDD binding to cytosol of beta-NF- or TCDD-treated rats yielded a small 4 S and a larger 8 S peak; only the latter was abolished by co-incubation with TCDF. Thus, the patterns of sedimentation, distribution and elimination of radioactivity from the 8 S fraction of the liver cytosols from beta-NF-, 3-MC-, TCDD- or alpha-NF-treated rats were characteristic for the AhR, whereas those from the 4 S fraction appeared specific for [(3)H]beta-NF binding. The data indicate that potent AhR agonists, TCDD, 3-MC and beta-NF, and to a lesser extent alpha-NF, a weak AhR agonist, induce a 4 S [(3)H]beta-NF-binding protein in liver cytosol of female rats. alpha-NF, beta-NF and 3-MC were effective competitors (80-85% inhibition) of the [(3)H]beta-NF-specific binding to the beta-NF-, 3 MC- or TCDD-induced 4 S protein, whereas several PAHs including B[a]P and benzo[e]pyrene were only weak competitors. The increased [(3)H]beta-NF binding was not associated with glycine N-methyltransferase activity. Hence, the 4 S [(3)H]beta-NF-binding protein described herein differs from the constitutive 4 S PAH-binding protein of rat liver cytosols in the inducibility by beta-NF and 3-MC, ligand-binding characteristics, and lack of glycine N-methyltransferase activity. Gel filtration on Sephacryl of liver cytosols from beta-NF-treated rats indicated a molecular mass of approximately 42 kDa for [(3)H]beta-NF-bound protein and suggested that it was derived from a large mass component that before the radioligand binding was eluted with the void volume of the gel and sedimented in a 7 S fraction of the sucrose gradient. The [(3)H]beta-NF binding activity was not eluted with glutathione S-transferase Ya, aldehyde-3-dehydrogenase or DT-diaphorase [NAD(P)H: quinone oxidoreductase] activities, which are AhR-controlled and beta-NF-inducible. Further studies are needed to determine the identity and function of this novel protein which may be involved either directly or indirectly (as a carrier protein) in xenobiotic metabolism in vivo.
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PMID:A novel 4 S [3H]beta-naphthoflavone-binding protein in liver cytosol of female Sprague-Dawley rats treated with aryl hydrocarbon receptor agonists. 1076 84

Ferredoxin NADP(H) oxidoreductases (FNR) are flavoenzymes that catalyze the electron transfer between NADP(H) and a wide range of compounds including ferredoxins and bacterial flavodoxins. FNRs are classified into two major groups: plant- and vertebrate-type. Plant-type FNRs are implicated in photosynthesis and nitrogen fixation in plastids and photosynthetic bacteria, and were recently implicated in cell protection against reactive oxygen species (ROS). Vertebrate-type FNRs are mitochondrial enzymes implicated in steroid hormone biosynthesis in mammals and in Fe(+) uptake and metabolism in yeasts. We have cloned and sequenced a cDNA coding for the vertebrate-type Schistosoma mansoni FNR. Gel diaphorase activity and western blot assays demonstrated that SmFNR represented the major diaphorase activity of adult worms. An active recombinant SmFNR was expressed in Escherichia coli that made the bacteria tolerant to oxygen peroxide, cumene hydroperoxide and the superoxide-generating herbicide, methyl viologen (MV).
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PMID:Schistosoma mansoni ferredoxin NADP(H) oxidoreductase and its role in detoxification. 1238 48


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