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

---

Query: EC:1.6.5.2 (NQO1)
6,196 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

This new assay procedure for diaphorase eliminates problems of high blank rates and nonlinear kinetics associated with other methods. The dye thiazolyl blue tetrazolium bromide is reduced in the presence of NADH and diaphorase to yield a colored formazan, which as maximum absorbance at 560 nm.
...
PMID:A new assay for diaphorase activity in reagent formulations, based on the reduction of thiazolyl blue. 4 50

Glutathione (GSH) has been shown to modulate the cytotoxicity of a variety of chemotherapeutic agents. The effect of mitomycin C (MMC) treatment duration and the effect of GSH depletion on in vitro cytotoxicity against the human colon cancer cell line HT-29 was studied under aerobic conditions. Continuous-exposure experiments revealed that the cytotoxicity of 0.1 microM MMC, as measured by clonogenic cell survival, exhibited a shoulder until exposure time was at least 12 h, after which time exponential cytotoxicity was observed. Lowering GSH levels to less than 3% of control using buthionine sulfoximine (BSO) did not enhance cytotoxicity of MMC given for 1 h or continuously for less than 12 h. However, GSH depletion did enhance cytotoxicity of MMC given continuously for at least 12 h, with a dose-modifying factor at 1% survival of 1.4 for a 24-h treatment. GSH depletion under these conditions enhanced cytotoxicity of even minimally cytotoxic MMC concentrations (0.02 microM). Absolute levels of GSH-related enzymes, including glutathione-S-transferase, and the MMC-metabolizing enzyme DT-diaphorase did not change appreciably. A tetrazolium [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay was used to verify the results further and to determine the optimal sequence of BSO administration with a 24-h MMC treatment. BSO added simultaneously with MMC did not increase cytotoxicity, compared to MMC alone. BSO added and then removed prior to MMC was effective (dose-modifying factor at 50% survival = 1.3), but the greatest cytotoxicity was noted when BSO was present before and during MMC treatment (dose-modifying factor = 1.5). GSH depletion in another cell line (SW480) showed similar enhancement of 24-h MMC cytotoxicity. These studies show that aerobic cytotoxicity of MMC is improved by administration of the drug in continuous fashion for at least 12 h, as opposed to continuous administration for shorter periods or 1-h bolus administration. Cytotoxicity of continuous (at least 12-h) MMC treatment can be modestly enhanced by GSH depletion, which must precede MMC exposure in order to be effective.
...
PMID:Effect of treatment duration and glutathione depletion on mitomycin C cytotoxicity in vitro. 151 28

We have determined the nucleotide sequence of a cDNA clone, pDTD55, complementary to rat liver quinone reductase mRNA (Williams, J.B., Lu, A.Y.H., Cameron, R.G., and Pickett, C.B. (1986) J. Biol. Chem. 261, 5524-5528). The cDNA clone contains an open reading frame of 759 nucleotides encoding a polypeptide comprised of 253 amino acids with a Mr = 28,564. To verify the predicted amino acid sequence of quinone reductase, we have been able to align the amino acid sequences of a cyanogen bromide digest of the purified enzyme to the sequence deduced from the cDNA clone. A comparison of the quinone reductase sequence with other known flavoenzymes did not reveal a significant degree of amino acid sequence homology. These data suggest that the quinone reductase gene has evolved independently from genes encoding other flavoenzymes.
...
PMID:Rat liver NAD(P)H: quinone reductase nucleotide sequence analysis of a quinone reductase cDNA clone and prediction of the amino acid sequence of the corresponding protein. 310 May 15

We describe a rapid and direct assay of NAD(P)H:(quinone-acceptor) oxidoreductase (EC 1.6.99.2) activity in cultured cells suitable for identifying and purifying inducers of this detoxication enzyme. Hepa 1c1c7 murine hepatoma cells are plated in 96-well microtiter plates, grown for 24 h, and exposed to inducing agents for another 24 h. The cells are then lysed and quinone reductase activity is assayed by the addition of a reaction mixture containing an NADPH-generating system, menadione (2-methyl-1,4-naphthoquinone), and MTT [3-(4,-5-dimethylthiazo-2-yl)-2,5-diphenyltetrazolium bromide]. Quinone reductase catalyzes the reduction of menadione to menadiol by NADPH, and MTT is reduced nonenzymatically by menadiol resulting in the formation of a blue color which can be quantitated on a microtiter plate absorbance reader. The reaction is more than 90% dicoumarol inhibitable and menadione dependent. The results are comparable to those obtained by harvesting cells from larger plates, preparing cytosols, and carrying out spectrophotometric measurements.
...
PMID:Direct measurement of NAD(P)H:quinone reductase from cells cultured in microtiter wells: a screening assay for anticarcinogenic enzyme inducers. 338 6

Enzymes involved in reduction of methyl p-nitrobenzoate in Escherichia coli B/r were oxygen-insensitive and precipitated between 30 and 60% ammonium sulfate saturation from cell-free extracts of the strain. The reductases were resolved by DEAE-cellulose column chromatography into three enzymes, NADH-linked, NAD(P)H-linked and NADPH-linked ones. These enzymes were flavoprotein which could be inactivated by dialysis against 1 M potassium bromide and could be reactivated by FMN. The NADH-linked and NAD(P)H-linked reductases were sensitive to dicumarol and exhibited menadione reductase activities. Aromatic nitro compounds with electron-withdrawing p-substituents were easily reduced by the NAD(P)H-linked reductase.
...
PMID:Studies on bacterial nitroreductases. Enzymes involved in reduction of aromatic nitro compounds in Escherichia coli. 634 84

Microculture tetrazolium assays (MTAs) rely upon the bioreduction of tetrazolium salts to their intensely coloured formazans. Although these assays are being extensively used, the intracellular mechanisms responsible for the formazan production are not known. MTAs currently provide the basis for uniquely precise in vitro bioassays for human growth hormone (hGH) which use the Nb2 cells. We have compared two contrasting tetrazolium salts, namely 3-(4,5-dimethyl-thiazol-2-yl)- 2,5-diphenyltetrazolium bromide (MTT) and 5-(3-carboxymethoxyphenyl)-2-(4,5-dimethylthiazolyl)-3-(4-++ +sulfophenyl) tetrazolium, inner salt (MTS), in this system. An intermediate electron acceptor (IEA) is obligatory for the MTS- but not the MTT-bioassay. We report that inhibitors of DT-diaphorase abolished MTS- but not MTT-formazan production. We conclude that substitution of MTT with MTS/menadione resulted in formazan production via a different electron transfer pathway which is exclusively mediated by DT-diaphorase.
...
PMID:Growth hormone-responsive DT-diaphorase-mediated bioreduction of tetrazolium salts. 883 14

Oltipraz [5-(2-pyrazinyl)-4-methyl-1,2-dithiole-3-thione] is a synthetic dithiolethione with chemopreventive activity against carcinogen-induced neoplasia of liver, lung, and colon in several animal model systems. Protection from tumor formation is associated with elevation of Phase II enzymes, including glutathione (GSH) transferase and NAD(P)H:quinone oxidoreductase (DT-diaphorase) in experimental carcinogenesis models in vivo. To investigate the time and dose relationships of the pharmacological action of oltipraz and to develop a model for its investigation, a human colon adenocarcinoma HT29 cell line was primarily used. In this cell line, oltipraz resulted in increased activity of both GSH transferase and DT-diaphorase. At the maximum effective concentration (100 microM), the elevation of GSH transferase was 3-fold and that of DT-diaphorase was 2-fold. The optimal duration of oltipraz exposure to HT29 cells was 24 h, following which the peak in enzyme activity was observed at 24 h after removal of the drug, and activity had almost returned to control levels after 72 h in drug-free media. Steady-state mRNA levels for DT-diaphorase were observed to increase during the period of drug exposure and remained elevated, even as catalytic activities declined to control levels, suggesting additional mechanisms for control of the activity of this enzyme. More prolonged drug exposure was associated with less induction of the detoxication enzymes, prompting an investigation of the possible toxicity of oltipraz to these cells. Although the 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay revealed inhibition of proliferation (IC50, 100 microM oltipraz), a clonogenic assay demonstrated no loss of clonogenicity. Oltipraz is known to be extensively metabolized in many species; two major metabolites include a 3-ketone (metabolite 2, M2) and a molecular rearrangement to a pyrrolopyrazine derivative (metabolite 3, M3), numerous conjugates of which are formed in vivo. To investigate the potential cause of the lag in response, we synthesized two major oltipraz metabolites (M2 and M3) and tested their efficacy in enzyme induction. The activity of DT-diaphorase was induced similarly by both oltipraz and M2 (2.6- versus 2.8-fold baseline) at 100 microM, whereas M3 was inactive at all concentrations. M2 also resulted in a 5.8-fold elevation of steady-state DT-diaphorase mRNA levels. Both enzyme activity and steady-state mRNA peaked at 24 h as with the parent compound. Thus, the oxidative desulfuration of oltipraz results in the formation of an active metabolite, but this process is not rate limiting for the induction of detoxicating enzymes. These data support the use of intermittent schedules in oltipraz in clinical trials of chemoprevention because of evidence of attenuation of response. The metabolite M2, but not M3, is as active as the parent compound and may be considered for clinical development in its own right.
...
PMID:Cellular kinetics of induction by oltipraz and its keto derivative of detoxication enzymes in human colon adenocarcinoma cells. 981 50

The human colon carcinoma cell lines Caco-2 and HT-29 were exposed to three structurally related naphthoquinones. Menadione (MEN), 1,4-naphthoquinone (NQ), and 2,3-dimethoxy-1,4-naphthoquinone (DIM) redoxcycle at similar rates, NQ is a stronger arylator than MEN, and DIM does not arylate thiols. The Caco-2 cell line was particularly vulnerable to NQ and MEN and displayed moderate toxic effects of DIM. The HT-29 cell line was only vulnerable to NQ and MEN after inhibition of DT-diaphorase (DTD) with dicoumarol, whereas dicoumarol did not affect the toxicity of quinones to Caco-2 cells. DTD activity in the HT-29 and Caco-2 cell lines, as estimated by the dicoumarol-sensitive reduction of 2,6-dichlorophenolindophenol, was 393.7 +/- 46.9 and 6.4 +/- 2.2 nmol NADPH x min(-1) x mg protein(-1), respectively. MEN depleted glutathione to a small extent in the HT-29 cell line, but a rapid depletion similar to Caco-2 cells was achieved when dicoumarol was added. The data demonstrated that the DTD-deficient Caco-2 cell line was more vulnerable to arylating or redoxcycling quinones than DTD-expressing cell lines. Exposure of the Caco-2 cell line to quinones produced a rapid rise in protein disulphides and oxidised glutathione. In contrast to NQ and DIM, no intracellular GSSG was observed with MEN. The relatively higher levels of ATP in MEN-exposed cells may account for the efficient extrusion of intracellular GSSG. The reductive potential of the cell as measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide reduction was only increased by MEN and not with NQ and DIM. We conclude that arylation is a major contributing factor in the toxicity of quinones. For this reason, NQ was the most toxic quinone, followed by MEN, and the pure redoxcycler DIM elicited modest toxicity in Caco-2 cells.
...
PMID:Quinone toxicity in DT-diaphorase-efficient and -deficient colon carcinoma cell lines. 992 Feb 82

Dihydrolipoamide dehydrogenase (LADH) lipoamide reductase activity decreased whereas enzyme diaphorase activity increased after LADH treatment with myeloperoxidase (MPO) dependent systems (MPO/H2O2/halide, MPO/NADH/halide and MPO/H2O2/nitrite systems. LADH inactivation was a function of the composition of the inactivating system and the incubation time. Chloride, iodide, bromide, and the thiocyanate anions were effective complements of the MPO/H2O2 system. NaOCl inactivated LADH, thus supporting hypochlorous acid (HOCl) as putative agent of the MPO/H2O2/NaCl system. NaOCl and the MPO/H2O2/NaCl system oxidized LADH thiols and NaOCl also oxidized LADH methionine and tyrosine residues. LADH inactivation by the MPO/NADH/halide systems was prevented by catalase and enhanced by superoxide dismutase, in close agreement with H2O2 production by the LADH/NADH system. Similar effects were obtained with lactoperoxidase and horse-radish peroxidase supplemented systems. L-cysteine, N-acetylcysteine, penicillamine, N-(2-mercaptopropionylglycine), Captopril and taurine protected LADH against MPO systems and NaOCl. The effect of the MPO/H2O2/NaNO2 system was prevented by MPO inhibitors (sodium azide, isoniazid, salicylhydroxamic acid) and also by L-cysteine, L-methionine, L-tryptophan, L-tyrosine, L-histidine and reduced glutathione. The summarized observations support the hypothesis that peroxidase-generated "reactive species" oxidize essential thiol groups at LADH catalytic site.
...
PMID:Inactivation of myocardial dihydrolipoamide dehydrogenase by myeloperoxidase systems: effect of halides, nitrite and thiol compounds. 1019 78

Dihydrolipoamide dehydrogenase (LADH) from Trypanosoma cruzi was inactivated by treatment with myeloperoxidase (MPO)-dependent systems. With MPO/H2O2/NaCl, LADH lipoamide reductase and diaphorase activities significantly decreased as a function of incubation time. Iodide, bromide, thiocyanide and chloride effectively supplemented the MPO/H2O2 system, KI and NaCl being the most and the least effective supplements, respectively. LADH inactivation by MPO/H2O2/NaCl and by NaOCl was similarly prevented by thiol compounds such as GSH, L-cysteine, N-acetylcysteine, penicillamine and N-(2-mercaptopropionyl-glycine) in agreement with the role of HOCI in LADH inactivation by MPO/H2O2/NaCl. LADH was also inactivated by MPO/NADH/halide, MPO/H2O2/NaNO2 and MPO/NADH/NaNO2 systems. Catalase prevented the action of the NADH-dependent systems, thus supporting H2O2 production by NADH-supplemented LADH. MPO inhibitors (4-aminobenzoic acid hydrazide, and isoniazid), GSH, L-cysteine, L-methionine and L-tryptophan prevented LADH inactivation by MPO/H2O2/NaNO2. Other MPO systems inactivating LADH were (a) MPO/H2O2/chlorpromazine; (b) MPO/H2O2/monophenolic systems, including L-tyrosine, serotonin and acetaminophen and (c) MPO/H2O2/di- and polyphenolic systems, including norepinephrine, catechol, nordihydroguaiaretic acid, caffeic acid, quercetin and catechin. Comparison of the above effects and those previously reported with pig myocardial LADH indicates that both enzymes were similarly affected by the MPO-dependent systems, allowance being made for T. cruzi LADH diaphorase inactivation and the greater sensitivity of its LADH lipoamide reductase activity towards the MPO/H2O2/NaCl system and NaOCl.
...
PMID:Trypanosoma cruzi dihydrolipoamide dehydrogenase is inactivated by myeloperoxidase-generated "reactive species". 1082 17


1 2 3 4 Next >>

---