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)

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

Inducibility of oxidative stress in rat liver in vivo by menadione-associated redox cycling activation under redox enzyme modulating conditions was examined by monitoring hepatocyte injury and 8-hydroxydeoxyguanosine (8-OHdG) levels of liver DNA. In addition, the treatment-associated liver tumor initiating activity was assessed in terms of development of gamma-glutamyl-transpeptidase (GGT)- and glutathione S-transferase placental form (GST-P)-positive foci and hyperplastic nodules. With or without following menadione treatment (50 mg/kg, i.g.), redox enzyme modulations of increased cytochrome P450 reductase activity induced by phenobarbital (PB)-Na (100 mg/kg, i.p. for 5 days), inhibition of DT-diaphorase by dicumarol (25 mg/kg, i.p.) and depletion of glutathione by phorone (200 mg/kg, i.p.), with or without further supplement of iron EDTA-Na-Fe(III) (70 mg/kg, i.p.), caused both substantial hepatocyte necrosis and 8-OHdG production in Fischer 344 male rats. Subsequent feeding with a 0.05% PB diet for 64 weeks resulted in slightly increased development of GGT-positive foci but not GST-P positive lesions or hyperplastic nodules, suggesting a lack of tumor-initiating activity of the oxidative DNA damage associated with redox enzyme modulations with or without menadione.
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PMID:Induction of 8-hydroxydeoxyguanosine but not initiation of carcinogenesis by redox enzyme modulations with or without menadione in rat liver. 170 52

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

A polymerase chain reaction (PCR)-based method was used to quantitate the expression levels of low abundance genes relevant to cancer drug activity. RNA from tumor samples as small as 20 mg was isolated and converted to cDNA using random hexamers. The 5' primers for the PCR contained a T7 polymerase promoter sequence, allowing the PCR-amplified DNA to be transcribed to RNA fragments. In each sample, the linear ranges of amplification of each cDNA of interest were established. Relative gene expressions were calculated by extrapolating the amounts of PCR products generated within the linear amplification regions of each gene to equal volumes of the cDNA solution. The method was accurate to less than a 2-fold difference in expression levels. Using beta 2-microglobulin and beta-actin gene expressions as internal reference standards and cDNA from HT-29 cells as an external linearity standard, we measured the relative expressions of thymidylate synthase, dihydrofolate reductase, and DT-diaphorase in a number of clinical tumor samples. The expressions of these genes varied from 50- to 100-fold among different tumors, although most of the values were grouped within about a 10-fold range. The amount of thymidylate synthase gene expression in tumor tissues was directly proportional to the content of thymidylate synthase protein. Those tumors with the lowest thymidylate synthase expression had the best response to both the 5-fluorouracil-leucovorin and 5-fluorouracil-cisplatin combinations.
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PMID:Quantitation of thymidylate synthase, dihydrofolate reductase, and DT-diaphorase gene expression in human tumors using the polymerase chain reaction. 172 69

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

Biotransformation in carcinogen-induced diploid and polyploid hepatocytes was studied using isozyme-selective substrates for several enzyme pathways. Diploid hepatocytes were induced by partial hepatectomy, a single injection of diethylnitrosamine, and 4 weeks of 2-acetylaminofluorene (2-AAF) feeding. Then, after an additional 3-5 weeks on the control diet, diploid and polyploid hepatocytes were separated from freshly isolated hepatocytes by centrifugal elutriation. Benzo(a)pyrene hydroxylase, ethoxyresorufin O-deethylase, and methoxycoumarin O-demethylase activities were approximately 15-40% lower in the diploid hepatocyte fraction than in the polyploid cell fraction. Activities of 1-chloro-2,4-dinitrobenzene, glutathione S-transferase, 3-hydroxy-benzo(a)pyrene or 4-hydroxybiphenyl UDP-glucuronosyltransferase, and DT-diaphorase were not different in the two cell fractions. Determination of activity during the 2-AAF treatment indicated that 2-AAF increased 7-ethoxyresorufin O-deethylase and 3-hydroxybenzo(a)pyrene glucuronosyltransferase activities by 300 and 200%, respectively, in both the diploid and polyploid hepatocyte fractions. Administration of phenobarbital for 4 days at the end of the control diet period increased ethoxyresorufin and methoxycoumarin dealkylations by 2- and 4-fold, and 3-hydroxybenzo(a)pyrene glucuronidation and 1-chloro-2,4-dinitrobenzene conjugation with glutathione by 1.5- to 2-fold in both hepatocyte fractions. Slight increases in benzo(a)pyrene hydroxylation and 4-hydroxybiphenyl glucuronidation were also evident in diploid cells. Although there is a slight decrease in cytochrome P-450-dependent monooxygenase activities, these data indicate that carcinogen-induced diploid hepatocytes do not show the typical toxicant-resistant phenotype observed in preneoplastic hepatocytes of altered liver foci, which are characterized by large decreases in monooxygenase biotransformations as well as increased activities of several phase II enzymes. This finding is compatible with the hypothesis that 2-AAF-induced nonploidizing growth of diploid hepatocytes is caused by nontoxic mechanisms in the present experimental paradigm. In addition, carcinogen-induced diploid cells respond to phenobarbital in a manner similar to that of polyploid hepatocytes.
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PMID:Biotransformation in carcinogen-induced diploid and polyploid hepatocytes separated by centrifugal elutriation. 173 74

3-Amino-1,2,4-benzotriazine-1,4-dioxide (SR 4233; WIN 59075) is a highly selective hypoxic cell cytotoxin soon to enter phase I clinical trial. The compound is thought to exert its action through a toxic one-electron reduced free radical intermediate. Preliminary data have suggested that SR 4233 may be metabolized by DT-diaphorase [NAD(P)H: (quinone acceptor) oxidoreductase (EC 1.6.99.2)] to both two- and four-electron reduced products and that this route of biotransformation may represent a bioprotection pathway. In this study, a highly purified enzyme preparation was employed in order to investigate further the metabolism of SR 4233 by DT-diaphorase and to examine the mechanism of reduction in more detail. Spectrophotometric analysis showed that SR 4233 underwent reduction by DT-diaphorase with an apparent Km of 1.23 +/- 0.27 mM and Vmax of 8.55 +/- 1.67 nmol/min/microgram protein. This reaction was inhibited completely by dicoumarol (100 microM) and partially by an antiserum raised against the purified enzyme. Characterization of the products of SR 4233 reduction by reverse-phase HPLC confirmed that both two- (SR 4317) and four- (SR 4330) electron reduction products were generated, the latter being the predominant metabolite, particularly in prolonged incubations. Further experiments showed that the four-electron reduction product, but not the two-electron reduction product, was also a substrate for DT-diaphorase with an apparent Km of 1.14 mM and a Vmax of 57.12 nmol/min/micrograms protein. The results presented confirm that SR 4233 is indeed a substrate for DT-diaphorase and that a mixture of two-, four- and six-electron reduced products may be formed. The possible toxicological and pharmacodynamic significance of this metabolism is discussed.
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PMID:Enzymology of the reduction of the potent benzotriazine-di-N-oxide hypoxic cell cytotoxin SR 4233 (WIN 59075) by NAD(P)H: (quinone acceptor) oxidoreductase (EC 1.6.99.2) purified from Walker 256 rat tumour cells. 173 5

A nonhepatic vitamin K-dependent protein, matrix Gla protein, has recently been identified in cartilage where it may play an important role in the control of mineralization or matrix development. We have investigated the vitamin K cycle in chondrocytes isolated from bovine and rabbit articular cartilage and examined these cells for their ability to synthesize vitamin K-dependent proteins. Chondrocytes were found to have an active vitamin K-dependent carboxylation system. Preincubation of the cells with warfarin resulted in a significant increase in the measured carboxylase activity. Both vitamin K epoxide reductase and DT-diaphorase (EC 1.6.99.2) activity were present indicating that chondrocytes are capable of producing reduced vitamin K1H2, the cofactor for the vitamin K-dependent carboxylase. Specific 14C-labeling of microsomal vitamin K-dependent protein precursors demonstrated synthesis of several vitamin K-dependent proteins by chondrocytes. 35S-labeling of chondrocyte proteins provided evidence that matrix Gla protein is synthesized by these cells.
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PMID:Vitamin K-dependent carboxylation in articular chondrocytes. 176 34

The effect of highly purified rat liver cytosolic NAD(P)H-quinone oxidoreductase [EC 1.6.99.2] on the mutagenicity of 1,3- 1,6- and 1,8-dinitropyrene (DNP) was studied in the Ames Salmonella typhimurium mutagenicity assay. NAD(P)H-quinone oxidoreductase over the range of 0.02-0.8 micrograms/plate (38-1500) units increased up to threefold the mutagenicity of all three DNPs in S. typhimurium TA 98. In TA98NR, a strain deficient in "classical" nitro-reductase, the mutagenicity of 1,6- and 1,8-DNP was essentially unchanged, whereas that of 1,3-DNP was markedly reduced. NAD(P)H-quinone oxidoreductase enhanced the mutagenicity of 1,6- and 1,8-DNP to approximately equivalent extents in TA98NR and TA98. The mutagenicity of 1,3-DNP in TA98NR was potently enhanced by the addition of NAD(P)H-quinone oxidoreductase in a dose-responsive manner. In the presence of 0.8 micrograms NAD(P)H-quinone oxidoreductase, 1,3-DNP displayed a mutagenic response in TA98NR that was comparable to that obtained in TA98. NAD(P)H-quinone oxidoreductase was found to increase the mutagenicity of 1,6- but not 1,3- or 1,8-DNP to mutagenic intermediates in TA98/1,8-DNP6, a strain deficient in O-acetyltransferase activity. The results suggest that NAD(P)H-quinone oxidoreductase not only catalyzes reduction of the parent DNP but also that of partially reduced metabolites generated from that DNP. Such reductive metabolism may lead to increased formation of the penultimate mutagenic species.
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PMID:Purified NAD(P)H-quinone oxidoreductase enhances the mutagenicity of dinitropyrenes in vitro. 177 70


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