Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:1.8.1.4 (diaphorase)
 2,754 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

NAD(P)H: quinone oxidoreductase (NQO1) is believed to be protective against cancer and toxicity caused by exposure to quinones and their metabolic precursors. This enzyme catalyzes the two-electron reduction of compounds, compared with one-electron reduction mediated by NADPH: cytochrome-P450 oxidoreductase which produces toxic and mutagenic free radicals. Recently we cloned and sequenced the cDNA encoding human 2.3,7,8-tetrachlorodibenzo-p-dioxin (dioxin)-inducible cytosolic NQO1 [Jaiswal et al. (1988) J. Biol. Chem. 263, 13572-13578] and provided preliminary evidence that this enzyme may correspond to diaphorase 4, an enzymatic activity present in various tissues that catalyzes the reduction of a variety of quinones by both NADH and NADPH [Edwards et al. (1980) Biochem. J. 187, 429-436]. In the present report we characterize the catalytic properties of the protein encoded by the NQO1 cDNA. The enzyme was synthesized in monkey kidney COS-1 cells transfected with a pMT2-based expression plasmid containing the NQO1 cDNA. Western blot analysis of the transfected cells using an antibody against rat liver cytosolic NQO1 revealed a 31-kDa band that was not detected in nontransfected cells. This band corresponded to a polypeptide with the same electrophoretic mobility as the endogenous NQO1 protein detected in the human hepatoblastoma (Hep-G2) cells with the same antibody. The immunoreactive protein detected in human Hep-G2 cells was induced approximately fourfold by exposure of the cultures to dioxin, an increase commensurate with the increased in quinone oxidoreductase activity. These studies suggest that the protein encoded by NQO1 cDNA is indeed similar, if not identical, to the dioxin-inducible protein band detected in human Hep-G2 cells. Further characterization of the product of NQO1 cDNA, which was present at approximately 20-30-fold higher levels in transfected COS cells than the endogenous product in uninduced human Hep-G2 cells indicated that it had very high capacity (greater than 1000-fold over background) to catalyze the reduction of 2.6-dichloroindophenol and menadione. Besides these two commonly used substrates for quinone reductase, the expressed NQO1 protein also effectively metabolized 2,6-dimethylbenzoquinone, methylene blue, p-benzoquinone, 1,4-naphthoquinone, 2-methyl-1,4-benzoquinone, with the latter being the most potent electron acceptor at 50 microM concentration of the substrate.
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PMID:The human dioxin-inducible NAD(P)H: quinone oxidoreductase cDNA-encoded protein expressed in COS-1 cells is identical to diaphorase 4. 189 80

Monkey kidney COS1 cells transiently transfected with plasmids pMT2-cytochrome P450 1A1 (CYP1A1), pMT2-cytochrome P450 reductase (P450 reductase), and pMT2-NAD(P)H:quinone oxidoreductase1 (NQO1 or DT diaphorase), individually or in combination, expressed significantly elevated levels of the respective enzyme(s). The transfected cells were homogenized to break cell membranes without affecting the nuclei and incubated with benzo[a]pyrene (BP) to determine the role of cDNA-encoded enzymes in metabolic activation and/or detoxification of BP. These studies were performed by measuring the capacity of the transfected cells to form DNA adducts as determined by 32P postlabeling and protein adduct detection. Cotransfection of the COS1 cells with cDNAs encoding CYP1A1 and P450 reductase resulted in eight distinct BP-DNA adducts. Inclusion of cDNA encoding NQO1 along with CYP1A1 and P450 reductase in transfection reduced the number of DNA adducts to six. The two lost DNA adducts were specifically eliminated due to the presence of cDNA-derived NQO1 activity. Subsequent experiments with BP-1,6-quinone, BP-3,6-quinone, and BP-6,12-quinone identified these two adducts as those of BP quinones. In an in vitro system, BP-3,6-quinone produced two adducts with deoxyguanosine (dG) but not with dA, dC, and dT. Furthermore, the positions of BP-3,6-quinone-dG adducts on TLC plate correspond to those that are prevented by cDNA-derived NQO1, thus identifying these adducts as BP quinones of dG. In addition, NQO1 reduced the amount of protein-BP adducts generated by CYP1A1 and P450 reductase into transfected COS1 cells. These results show that semiquinones can directly bind to DNA and demonstrate that NQO1 activity can specifically reduce the binding of quinone metabolites of BP generated by CYP1A1 and P450 reductase to DNA and protein.
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PMID:NAD(P)H:quinone oxidoreductase1 (DT diaphorase) specifically prevents the formation of benzo[a]pyrene quinone-DNA adducts generated by cytochrome P4501A1 and P450 reductase. 807 96

The geno- and cytotoxicity of chromate, an important environmental pollutant, is partly attributed to the flavoenzyme-catalyzed reduction with the concomitant formation of reactive oxygen species. The aim of this work was to characterize the role of NAD(P)H:quinone oxidoreductase (NQO1, DT-diaphorase, EC 1.6.99.2) and glutathione reductase (GR, EC 1.6.4.2) in the mammalian cell cytotoxicity of chromate, which was evidenced controversially so far. The chromate reductase activity of NQO1 was higher than that of GR, but lower than that of lipoamide dehydrogenase (EC 1.6.4.3), ferredoxin:NADP+ reductase (EC 1.18.1.2), and NADPH: cytochrome P-450 reductase (EC 1.6.2.4). The reduction of chromate by NQO1 was accompanied by the formation of reactive oxygen species. The concentration of chromate for 50% survival of bovine leukemia virus-transformed lamb kidney fibroblasts (line FLK) during a 24-h incubation was (22 +/- 4) microM. The cytotoxicity was partly prevented by desferrioxamine, the antioxidant N,N'-diphenyl-p-phenylene diamine and by an inhibitor of NQO1, dicumarol, and potentiated by 1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU), which inactivates GR. The NADPH-dependent chromate reduction by digitonin-permeabilized FLK cells was partly inhibited by dicumarol and not affected by BCNU. Taken together, these data indicate that the oxidative stress-type cytotoxicity of chromate in FLK cells may be partly attributed to its reduction by NQO1, but not by GR. The effect of BCNU on the chromate cytotoxicity may indicate that the general antioxidant action of reduced glutathione is more important than its prooxidant activities arising from the reactions with chromate.
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PMID:Prooxidant cytotoxicity of chromate in mammalian cells: the opposite roles of DT-diaphorase and glutathione reductase. 1729 2

Although the lung is one of the least studied organs in diabetes, increasing evidence indicates that it is an inevitable target of diabetic complications. Nevertheless, the underlying biochemical mechanisms of lung injury in diabetes remain largely unexplored. Given that redox imbalance, oxidative stress, and mitochondrial dysfunction have been implicated in diabetic tissue injury, we set out to investigate mechanisms of lung injury in diabetes. The objective of this study was to evaluate NADH/NAD+ redox status, oxidative stress, and mitochondrial abnormalities in the diabetic lung. Using STZ induced diabetes in rat as a model, we measured redox-imbalance related parameters including aldose reductase activity, level of poly ADP ribose polymerase (PAPR-1), NAD+ content, NADPH content, reduced form of glutathione (GSH), and glucose 6-phophate dehydrogenase (G6PD) activity. For assessment of mitochondrial abnormalities in the diabetic lung, we measured the activities of mitochondrial electron transport chain complexes I to IV and complex V as well as dihydrolipoamide dehydrogenase (DLDH) content and activity. We also measured the protein content of NAD+ dependent enzymes such as sirtuin3 (sirt3) and NAD(P)H: quinone oxidoreductase 1 (NQO1). Our results demonstrate that NADH/NAD+ redox imbalance occurs in the diabetic lung. This redox imbalance upregulates the activities of complexes I to IV, but not complex V; and this upregulation is likely the source of increased mitochondrial ROS production, oxidative stress, and cell death in the diabetic lung. These results, together with the findings that the protein contents of DLDH, sirt3, and NQO1 all are decreased in the diabetic lung, demonstrate that redox imbalance, mitochondrial abnormality, and oxidative stress contribute to lung injury in diabetes.
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PMID:Redox imbalance and mitochondrial abnormalities in the diabetic lung. 2788 91

The objective of this study was to investigate a possible role of mitochondrial dihydrolipoamide dehydrogenase (DLDH) as a chemical preconditioning target for neuroprotection against ischemic injury. We used 5-methoxyindole-2-carboxylic acid (MICA), a reportedly reversible DLDH inhibitor, as the preconditioning agent and administered MICA to rats mainly via dietary intake. Upon completion of 4 week's MICA treatment, rats underwent 1h transient ischemia and 24h reperfusion followed by tissue collection. Our results show that MICA protected the brain against ischemic stroke injury as the infarction volume of the brain from the MICA-treated group was significantly smaller than that from the control group. Data were then collected without or with stroke surgery following MICA feeding. It was found that in the absence of stroke following MICA feeding, DLDH activity was lower in the MICA treated group than in the control group, and this decreased activity could be partly due to DLDH protein sulfenation. Moreover, DLDH inhibition by MICA was also found to upregulate the expression of NAD(P)H-ubiquinone oxidoreductase 1(NQO1) via the Nrf2 signaling pathway. In the presence of stroke following MICA feeding, decreased DLDH activity and increased Nrf2 signaling were also observed along with increased NQO1 activity, decreased oxidative stress, decreased cell death, and increased mitochondrial ATP output. We also found that MICA had a delayed preconditioning effect four weeks post MICA treatment. Our study indicates that administration of MICA confers chemical preconditioning and neuroprotection against ischemic stroke injury.
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PMID:Administration of 5-methoxyindole-2-carboxylic acid that potentially targets mitochondrial dihydrolipoamide dehydrogenase confers cerebral preconditioning against ischemic stroke injury. 2901 57

Provision of NAD+ for oxidative decarboxylation of alpha-ketoglutarate to succinyl-CoA by the ketoglutarate dehydrogenase complex (KGDHC) is critical for maintained operation of succinyl-CoA ligase yielding high-energy phosphates, a process known as mitochondrial substrate-level phosphorylation (mSLP). We have shown previously that when NADH oxidation by complex I is inhibited by rotenone or anoxia, mitochondrial diaphorases yield NAD+, provided that suitable quinones are present (Kiss G et al., FASEB J 2014, 28:1682). This allows for KGDHC reaction to proceed and as an extension of this, mSLP. NAD(P)H quinone oxidoreductase 1 (NQO1) is an enzyme exhibiting diaphorase activity. Here, by using Nqo1-/- and WT littermate mice we show that in rotenone-treated, isolated liver mitochondria 2-methoxy-1,4-naphtoquinone (MNQ) is preferentially reduced by matrix Nqo1 yielding NAD+ to KGDHC, supporting mSLP. This process was sensitive to inhibition by specific diaphorase inhibitors. Reduction of idebenone and its analogues MRQ-20 and MRQ-56, menadione, mitoquinone and duroquinone were unaffected by genetic disruption of the Nqo1 gene. The results allow for the conclusions that i) MNQ is a Nqo1-preferred substrate, and ii) in the presence of suitable quinones, mitochondrially-localized diaphorases other than Nqo1 support NADH oxidation when complex I is inhibited. Our work confirms that complex I bypass can occur by quinones reduced by intramitochondrial diaphorases oxidizing NADH, ultimately supporting mSLP. Finally, it may help to elucidate structure-activity relationships of redox-active quinones with diaphorase enzymes.
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PMID:Reduction of 2-methoxy-1,4-naphtoquinone by mitochondrially-localized Nqo1 yielding NAD+ supports substrate-level phosphorylation during respiratory inhibition. 2974 24