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 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

Coenzyme Q (CoQ0) and other quinones were shown to be potent insulin secretagogues in the isolated pancreatic islet. The order of potency was CoQ0 congruent to benzoquinone congruent to hydroquinone-menadione. CoQ6 and CoQ10 (ubiquinone), duroquinone and durohydroquinone did not stimulate insulin release. CoQ0's insulinotropism was enhanced in calcium-free medium and CoQ0 appeared to stimulate only the second phase of insulin release. CoQ0 inhibited inositol mono-, bis- and trisphosphate formation. Inhibitors of mitochondrial respiration (rotenone, antimycin A, FCCP and cyanide) and the calcium channel blocker verapamil, did not inhibit CoQ0-induced insulin release. Dicumarol, an inhibitor of quinone reductase, did not inhibit CoQ0-induced insulin release, but it did inhibit glucose-induced insulin release suggesting that the enzyme and quinones play a role in glucose-induced insulin release. Quinones may stimulate insulin release by mimicking physiologically-occurring quinones, such as CoQ10, by acting on the plasma membrane or in the cytosol. Exogenous quinones may bypass the quinone reductase reaction, as well as many reactions important for exocytosis.
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PMID:Stimulation of insulin release from pancreatic islets by quinones. 172 Mar 33

Cibacron Blue, a widely used ligand for affinity chromatography, is a potent inhibitor of NAD(P)H:(quinone-acceptor) oxidoreductase (EC 1.6.99.2) (quinone reductase). This property has been exploited to purify quinone reductase, to identify its nucleotide-binding site, and to obtain diffraction-grade crystals of this enzyme [Prochaska, H. J. (1988) Arch. Biochem. Biophys. 267, 529-538; Ysern, X., & Prochaska, H. J. (1989) J. Biol. Chem. 264, 7765-7767]. To define the structural region(s) of the dye responsible for its inhibitory potency, Cibacron Blue was synthesized and the dye, its synthetic intermediates, and some analogues of these intermediates were crystallized as novel trialkylamine or choline salts. These compounds were characterized by proton NMR and mass spectrometry, and their inhibitory potencies were measured. Only two of the four ring systems of the Cibacron Blue molecule are required for potent inhibition. Acid Blue 25 [1-amino-4-(phenylamino)anthraquinone-2-sulfonic acid] is an inhibitor (Ki = 22 nM) almost as potent as Cibacron Blue (Ki = 6.2 nM). However, removal of any of the three substituents on the anthraquinone ring of Acid Blue 25 markedly reduced inhibitory potency. These results are consistent with the proposal that Cibacron Blue is primarily a mimic for the ADP fragment of mono- and dinucleotides. The difference absorption spectrum of the Acid Blue 25-quinone reductase complex was very different from that of the complex with Cibacron Blue. In contrast to other compounds tested, Procion Blue M-3GS, the electrophilic dichlorotriazine precursor of Cibacron Blue, was an irreversible inhibitor of quinone reductase (KD = 16 nM, k3 = 0.03 min-1), and the inactivation was blocked by Cibacron Blue, a monochlorotriazine.
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PMID:Inhibition of NAD(P)H:(quinone-acceptor) oxidoreductase by cibacron blue and related anthraquinone dyes: a structure-activity study. 173 40

NAD(P)H:quinone oxidoreductase (DT-diaphorase; DTD) is an obligate two-electron reductase which may play a role in the bioactivation of antitumor quinones such as mitomycin C (MMC). We studied 10 colon carcinoma cell lines showing different levels of DTD activity (range, 0-3447 nmol/min/mg protein), as measured by the reduction of dichlorophenolindophenol. Expression of the NAD(P)H:quinone reductase gene (NQO1), which codes for the DTD enzyme, as measured by a polymerase chain reaction amplification technique was then correlated with enzymatic activity in all cell lines. HT-29 cells, which have intermediate DTD activity (769 +/- 144 nmol/min/mg protein, mean +/- SD) and are sensitive to MMC, showed high NQO1 expression relative to beta-actin (taken as 100% here for comparative purposes). BE cells which have no detectable DTD activity and are resistant to MMC showed moderate NQO1 expression (91% of HT-29). RNA single-strand conformational polymorphism analysis and subsequent sequencing of BE complementary DNA revealed a C to T mutation in the NQO1 complementary DNA. This confers a proline to serine substitution in the amino acid sequence of the protein. Additionally, HCT-116 cells showed both moderate DTD activity (390 +/- 41 nmol/min/mg protein) and NQO1 expression (41% of HT-29), while resistant subclones of these cells, exposed to MMC during 11 and 44 weeks, showed low gene expression (5 and 9% of HT-29 respectively) and enzymatic activity (11 +/- 6 and 36 +/- 16 nmol/min/mg protein). These results support the ideas that reductive activation of MMC by DTD may be important in the cytotoxicity of MMC and that polymerase chain reaction may be a useful technique for quantitating the relative expression of genes in human tumors.
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PMID:NAD(P)H:quinone oxidoreductase gene expression in human colon carcinoma cells: characterization of a mutation which modulates DT-diaphorase activity and mitomycin sensitivity. 173 39

The effect of incubating young placental explants with HgCl2 on the activities of aryl hydrocarbon hydroxylase (AHH) (a phase I enzyme), quinone reductase (QR), catecholamine-O-methyltransferase (COMT) (both phase II enzymes), and glucose-6-phosphate dehydrogenase (G-6-PD) is described. Mercury (Hg) at low doses significantly elevated placental phase I and phase II enzyme activities, but decreased the activity of G-6-PD. The increase in activities, which was time- and dose-dependent, was higher in explants incubated for 24 hr than in those incubated for 6 hr. The decrease in placental G-6-PD activity was drastic at low Hg dose levels but at higher levels the inhibitory effect was milder for both incubation periods. Placental explants accumulated Hg in amounts proportional to its concentration in the incubation medium and this accumulation was greater in explants incubated for 24 hr. The data suggest that contamination with low Hg levels from the environment during pregnancy may affect placental enzymatic activity. The accumulation of Hg during short incubation indicates a strong placental cell affinity for Hg, which could affect its other metabolic functions. The system used in sensitive, as it shows alteration in enzyme activity even with relatively low concentrations of the metal and the response is dose-related.
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PMID:In vitro effect of mercury on enzyme activities and its accumulation in the first-trimester human placenta. 174 99

The toxicity of quinones--including certain chemotherapeutic agents such as doxorubicin--have been related to the enzymatic or nonenzymatic formation of the corresponding semiquinones and their subsequent reaction with molecular oxygen yielding superoxide anion radicals by spontaneous regenerating of the quinones. This semiquinone redox cycling is prevented by the NAD(P)H:quinone reductase (NQR; EC 1.6.99.2) because it mediates a 2-electron reduction which results in the formation of hydroquinones instead of semiquinones. Interestingly, inducers of this enzyme such as butylated hydroxytoluene protect against the severe ulceration of accidental infiltration of doxorubicin into the area around the intravenous infusion. Recently, it has been shown that this highly protective enzyme has a very high basal activity in the epidermis which is in the same range as in the liver. The human gene of the NQR is localized on chromosome 16 and has been cloned recently as well as the gene of the murine liver NQR. We determined NQR in the cytoplasma of murine skin, liver, and human keratinocytes using 2,6-dichlorophenol-indophenol as substrate. In order to characterize this enzyme, induction by polycyclic hydrocarbones and inhibition with several known inhibitors of dihydrodiol dehydrogenase, aldo-keto and carbonyl reductase activities were determined. There was a similar pattern of inhibition of the basal and induced activity in all tissues so far investigated. Pyrazole, progesterone and phenobarbital did not inhibit; however, rutin and indomethacin inhibited dose-dependently. The most potent inhibitor was dicoumarol. These findings suggest that the same enzymatic form is present in liver and skin, and in murine skin and human keratinocytes.
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PMID:Cutaneous NAD(P)H: quinone reductase: a xenobiotica-metabolizing enzyme with potential cancer and oxidation stress-protecting properties. 176 53

The purpose of this study was to characterize the human cutaneous NAD(P)H: quinone reductase (NQR) activity by known inhibitors of different reductases and to compare it with the murine skin and liver NQR activity. This enzyme plays a major role in the defence of cells against oxygen stress because it inhibits the 1-electron reduction of quinones to semiquinones and their subsequent oxidation to quinones termed as quinone redox cycle. It belongs to the aromatic hydrocarbon-responsive (Ah) battery. This gene battery includes Cyp1a1 (cytochrome P-450 IA1), Cyp1a2 (cytochrome P-450 IA2) and Nmo-1 [NAD(P)H: quinone reductase]. In the skin cytochrome P-450 IA1-dependent activity is about 1-5% compared to the corresponding activity in the liver, whereas NQR has the same activity in skin and liver. NQR was determined in the cytoplasm of murine skin, liver, and human keratinocytes using 2,6-dichlorophenolindophenol as the substrate. The Ah-receptor binding compounds, such as coal tar constituents, or 3-methylcholanthrene induce cytochrome P-450-dependent activities such as aryl hydrocarbon hydroxylase or 7-ethoxyresorufin-O-de-ethylase and NQR, whereas butyl hydroxytoluol, which does not bind to the Ah receptor, induces only NQR. For inhibition studies several known inhibitors of dihydrodiol dehydrogenase, aldo-keto and carbonyl reductase activities were used. There was a similar pattern of inhibition of the basal and induced activity in all tissues investigated. Pyrazole, progesterone and phenobarbital did not inhibit, whereas dicoumarol, rutin and indomethacin inhibited NQR activity in murine skin and liver as well as in human keratinocytes.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Induction and inhibition of NAD(P)H: quinone reductase in murine and human skin. 176 30


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