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)

We have characterized further the antioxidant responsive element (ARE) identified in the 5'-flanking region of the rat glutathione S-transferase Ya subunit gene and the NAD(P)H:quinone reductase gene by mutational and deletion analyses. Our data suggest that the sequence, 5'-puGTGACNNNGC-3' 3'-pyCACTGNNNCG-5' where N is any nucleotide, represents the core sequence of the ARE required for transcriptional activation by phenolic antioxidants and metabolizable planar aromatic compounds (e.g. beta-naphthoflavone and 3-methylcholanthrene). We also have found that the ARE is responsive to hydrogen peroxide and phenolic antioxidants that undergo redox cycling. These latter data suggest that the ARE is responsive to reactive oxygen species and thus may represent part of a signal transduction pathway that allow eukaryotic cells to sense and respond to oxidative stress.
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PMID:The antioxidant responsive element. Activation by oxidative stress and identification of the DNA consensus sequence required for functional activity. 164 13

The induction of quinone reductase [QR; NAD(P)H:(quinone acceptor) oxidoreductase; EC 1.6.99.2] in cultured cells and animal tissues of rodents has provided useful information on mechanisms of protection against carcinogens. We have developed a simple and efficient microtiter plate assay for the direct measurement of QR basal activity and inducibility in human peripheral blood lymphocytes (unstimulated, mitogen-stimulated and Epstein-Barr virus-transformed) grown in suspension culture. In these cells, QR was induced by monofunctional (electrophilic) inducers (i.e. 1,2-dithiole-3-thione, dimethyl fumarate, methyl vinyl sulfone) but not by bifunctional inducers (i.e. 1,1'-azonaphthalene, beta-naphthoflavone, 2,3,7,8-tetrachlorodibenzo-p-dioxin). QR is a major enzyme of xenobiotic metabolism that carries out obligatory two-electron reductions and thereby protects cells against the toxicity of quinones. It is induced in many tissues coordinately with other enzymes that protect against electrophiles. Since lymphocytes can be sampled easily and repetitively in man, this system may provide a simple short-term marker for assessing the capacity of tissues to detoxify electrophiles, such as quinones, and for measuring the response to inducers.
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PMID:Induction of NAD(P)H:quinone reductase in human peripheral blood lymphocytes. 166 Jul 93

NAD(P)H:quinone oxidoreductases (NQOs) are flavoproteins that catalyze the oxidation of NADH or NADPH by various quinones and oxidation-reduction dyes. We have previously described a complementary DNA that encodes a dioxin-inducible cytosolic form of human NAD(P)H:quinone oxidoreductase (NQO1). In the present report we describe the nucleotide sequence and deduced amino acid sequence for a cDNA clone that is likely to encode a second form of NAD(P)H:quinone oxidoreductase (NQO2) which was isolated by screening a human liver cDNA library by hybridization with a NQO1 cDNA probe. The NQO2 cDNA is 976 nucleotides long and encodes a protein of 231 amino acids (Mr = 25,956). The human NQO2 cDNA and protein are 54% and 49% similar to human liver cytosolic NQO1 cDNA and protein, respectively. COS1 cells transfected with NQO2 cDNA showed a 5-7-fold increase in NAD(P)H:quinone oxidoreductase activity as compared to nontransfected cells when either 2,6-dichlorophenolindophenol or menadione was used as substrate. Western blot analysis of the expressed NQO1 and NQO2 cDNA proteins showed cross-reactivity with rat NQO1 antiserum, indicating that NQO1 and NQO2 proteins are immunologically related. Northern blot analysis shows the presence of one NQO2 mRNA of 1.2 kb in control and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) treated human hepatoblastoma Hep-G2 cells and that TCDD treatment does not lead to enhanced levels of NQO2 mRNA as it does for NQO1 mRNA. Southern blot analysis of human genomic DNA suggests the presence of a single gene approximately 14-17 kb in length. The NQO2 gene locus is highly polymorphic as indicated by several restriction fragment length polymorphisms detected with five different restriction enzymes.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Nucleotide and deduced amino acid sequence of a human cDNA (NQO2) corresponding to a second member of the NAD(P)H:quinone oxidoreductase gene family. Extensive polymorphism at the NQO2 gene locus on chromosome 6. 169 23

The glucuronide conjugates of oroxylin A and two other flavones, baicalein, and wogonin, were isolated from the methanol extract of the herb scutellariae radix (Huang Qin) and were found to be inhibitors of rat liver NAD(P)H:quinone acceptor oxidoreductase (EC 1.6.99.2). Baicalin (baicalein 7-O-glucuronide) and oroxylin-A 7-O-glucuronide are approximately 50-fold more potent than wogonin 7-O-glucuronide. The enzyme kinetic analysis revealed that oroxylin-A 7-O-glucuronide is a competitive inhibitor with respect to NADH (the electron donor), with a Ki value of 63 nM. Considering the similarities of their structures and inhibition kinetics to those of dicoumarol, it is thought that oroxylin-A 7-O-glucuronide and the other two flavonoids bind to an identical site and inhibit this quinone reductase in the same fashion as dicoumarol. The results also suggest that the inhibition of NAD(P)H:quinone acceptor oxidoreductase or another vitamin K reductase by oroxylin-A 7-O-glucuronide and the related flavonoids may be one of the steps associated with the anticoagulation action of the herb. These compounds are potentially useful anticoagulant drugs.
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PMID:Inhibition of rat liver NAD(P)H:quinone acceptor oxidoreductase (DT-diaphorase) by flavonoids isolated from the Chinese herb scutellariae radix (Huang Qin). 169 61

A sensitive enzyme immunoassay was developed for human angiotensin converting enzyme. Monoclonal antibodies specific for two unique converting enzyme epitopes were utilized to develop a two-site sandwich enzyme immunoassay. Alkaline phosphatase conjugated to the detecting antibody hydrolyzes nicotinamide adenine dinucleotide phosphate (NADP) to NAD. Subsequently, NAD is cycled between its reduced and oxidized forms by an alcohol dehydrogenase/diaphorase catalyzed redox cycle. Each cycle converts iodonitrotetrazolium violet to a highly colored formazan which is quantitated. With this assay, as little as 94 pg/ml of native converting enzyme is detectable without interference from either therapeutic or endogenous converting enzyme inhibitors.
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PMID:A sensitive two-site sandwich enzyme immunoassay for human angiotensin converting enzyme utilizing monoclonal antibodies. 169 77

A photoaffinity analog of 4-hydroxycoumarin containing an arylazido derivative at the 3-position has been synthesized and characterized. This compound, 3-(4-azido-5-iodosalicylamido)-4-hydroxycoumarin, serves as a strong competitive inhibitor of the dicoumarol-sensitive NAD(P)H: quinone reductase (DT-diaphorase) from rat liver, having an apparent inhibition constant of 4.2 10(-7) M. Irradiation of the reductase with ultraviolet light in the presence 10 microM of the photoprobe resulted in the covalent labeling of 2% of the reductase molecules. The enzyme is protected from labeling to greater than 99% by the inclusion of 3 microM dicoumarol, consistent with the specific labeling of the 4-hydroxycoumarin binding site of this enzyme. Furthermore, the quinone reductase was shown to specifically labeled by the probe even when contained within crude fractions rat liver cytosol.
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PMID:Synthesis of 3-(4-azido-5-iodosalicylamido)-4-hydroxycoumarin: photoaffinity labeling of rat liver dicoumarol-sensitive NAD(P)H: quinone reductase. 170 Jul 3

Glutamate toxicity in the N18-RE-105 neuronal cell line results from the inhibition of high-affinity cystine uptake, which leads to a depletion of glutathione and the accumulation of oxidants. Production of superoxides by one-electron oxidation/reduction of quinones is decreased by NAD(P)H:quinone reductase, an enzyme with DT-diaphorase activity. Using glutamate toxicity in N18-RE-105 cells as a model of neuronal oxidative stress, we report that the degree of glutamate toxicity observed is inversely proportional to quinone reductase activity. Induction of quinone reductase activity by treatment with t-butylhydroquinone reduced glutamate toxicity by up to 80%. In contrast, treatment with the quinone reductase inhibitor dicumarol potentiated the toxic effect of glutamate. Measurement of cellular glutathione indicates that increases in its levels are not responsible for the protective effect of t-butylhydroquinone treatment. Because many types of cell death may involve the formation of oxidants, induction of quinone reductase may be a new strategy to combat neurodegenerative disease.
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PMID:Enhanced NAD(P)H:quinone reductase activity prevents glutamate toxicity produced by oxidative stress. 170 27

A photoaffinity analog of 4-hydroxycoumarin containing an azidobenzyl group at the 3-position and, if desired, carbon-14 or tritium radionuclides has been synthesized and characterized. This compound, 3-(p-azidobenzyl)-4-hydroxycoumarin, serves as an effective competitive inhibitor of the dicoumarol-sensitive NAD(P)H:quinone reductase (EC 1.6.99.2; DT-diaphorase) from rat liver, having an apparent inhibition constant of 6.6 x 10(-8) M, a value comparable to that observed for dicoumarol (1.7 x 10(-9) M), significantly lower than for Warfarin (3.5 x 10(-5) M) and well within the range required of an effective photoaffinity reagent. Irradiation of the reductase with ultraviolet light in the presence of the photoprobe resulted in the covalent labeling of up to 10% of the protein. Greater than 99% of the covalent incorporation is precluded by the addition of 15 microM dicoumarol, consistent with the specific labeling of the 4-hydroxycoumarin binding site of this enzyme by this photoaffinity reagent. Further evidence of a high degree of specificity is provided by the isolation and sequence analysis of the peptides covalently modified by this reagent. A single region within the protein was found to be labeled, with threonine 127 and tyrosine 128 being the only amino acid residues that were observed to be modified. These results, for the first time, define a portion of the 4-hydroxycoumarin binding site within a protein that has a well established sensitivity to this type of anticoagulant and, because dicoumarol serves as a competitive inhibitor for pyridine nucleotides in this enzyme, may also define a portion of this unusual pyridine nucleotide binding site. In addition, these results suggest that this reagent may be effective as a highly specific photoaffinity probe in the identification of other proteins that are similarly inhibited by 4-hydroxycoumarin derivatives, such as the microsomal enzymes associated with the vitamin K-dependent carboxylation system.
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PMID:Synthesis of the photoaffinity probe 3-(p-azidobenzyl)-4-hydroxycoumarin and identification of the dicoumarol binding site in rat liver NAD(P)H:quinone reductase (EC 1.6.99.2). 170 34

EO9 [3-hydroxymethyl-5-aziridinyl-1-methyl-2-(H-indole-4, 7-indione)-propenol] is a novel indoloquinone structurally related to mitomycin C, a quinone anticancer drug that requires reductive bioactivation. NAD(P)H: (quinone-acceptor) oxidoreductase (quinone reductase, DT-diaphorase, EC 1.6.99.2) is an obligate 2-electron donating enzyme that can reduce a variety of quinones resulting either in bioactivation or bioprotection. Using quinone reductase (QR) preparations from rat Walker 256 mammary tumor cells and human HT29 colon carcinoma cells, we have characterized the role of this enzyme in EO9 reductive metabolism. QR activity was assayed under optimal conditions by following cytochrome c reduction at 550 nm in the presence of enzyme, quinone substrate, NADH, and bovine albumin, and confirmed by loss of EO9 absorbance at 550 nm. Both the rat and human tumor cell enzymes catalyzed reduction of the benchmark quinone menadione with a similar Km of 1.4-3.1 microM, although the Vmax was 7 to 8-fold lower for the human preparation. EO9 was readily reduced by the rat Walker QR. The mean Km was about 5-fold higher than for menadione at around 15 microM and the Vmax was 6-fold lower at around 2.5 mumol of cytochrome c reduced mg-1 of protein. EO9 was also metabolized by QR from HT29 human colon carcinoma cells but rather less efficiently than by the rat tumor enzyme. For example, the rate was 6-fold lower than that for the Walker tumor enzyme at 100 microM substrate concentration after correcting for the 7- to 8-fold difference in specific activity for the two preparations.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:The role of NAD(P)H: quinone reductase (EC 1.6.99.2, DT-diaphorase) in the reductive bioactivation of the novel indoloquinone antitumor agent EO9. 171 84

The participation of DT-diaphorase or NAD(P)H:(quinone acceptor) oxidoreductase (E.C. 1.6.99.2) in metabolism or in events leading to toxicity is often implied on the basis of the inhibitory effects of dicoumarol. DT-diaphorase functions via a ping pong bi-bi kinetic mechanism involving oxidized and reduced flavin forms of the free enzyme. Dicoumarol, a potent (Ki = 10 nM) inhibitor, binds to the oxidized form of the enzyme, competitively versus reduced pyridine nucleotide. Inhibition is effectively complete at 1 microM dicoumarol in typical studies using DCPIP, one of the best known substrates for the enzyme, as electron acceptor. The antitumor quinone Diaziquone (AZQ) is a poor substrate for DT-diaphorase relative to DCPIP, but effective inhibition of its reduction requires ten-fold higher concentrations of dicoumarol than for inhibition of DCPIP reduction under otherwise similar conditions. The variable inhibition of DT-diaphorase by dicoumarol dependent on the efficiency of the electron acceptor can be explained on the basis of the complete rate equation describing its ping pong type kinetic mechanism. Thus, the concentration of dicoumarol used to inhibit DT-diaphorase must be chosen carefully and consideration should be given to the efficiency of the electron acceptor. The absence of an inhibitory effect using low doses of dicoumarol cannot rule out a reaction mediated by DT-diaphorase. Although higher doses of dicoumarol may be required to inhibit DT-diaphorase mediated metabolism of less efficient electron acceptors, the use of such doses in cells may also affect biochemical processes other than DT-diaphorase and should be approached with caution.
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PMID:A note on the inhibition of DT-diaphorase by dicoumarol. 171 26


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