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 previously reported the expression of a full-length cDNA complementary to a rat liver NAD(P)H:quinone oxidoreductase (EC 1.6.99.2) mRNA in Escherichia coli (Q. Ma, R. Wang, C. S. Yang, and A. Y. H. Lu, 1990, Arch. Biochem. Biophys. 283, 311-317). Since cysteine residues have been suggested to be important for the catalysis of flavoproteins and a lysine residue at position 76 in NAD(P)H:quinone oxidoreductase has been proposed to be involved in electron transfer of the enzyme, we investigated the roles of lysine 76 and cysteine 179 of this enzyme in catalysis by site-directed mutagenesis. Mutant cDNA clones replacing lysine 76 with valine (K76V) and cysteine 179 with alanine (C179A) were generated by a procedure based on the polymerase chain reaction. The mutant enzymes were expressed in E. coli. The cytosolic activities of the K76V and C179A mutants were 50 and 25% of that of the wild type (DTD), due to lower levels of the mutant proteins as shown by immunoblot analysis. The mutant proteins were purified to apparent homogeneity. The purified K76V and C179A mutant enzymes maintained full activities of 2,6-dichlorophenolindophenol (DCIP) reduction compared with that of the wild type. The mutant enzymes exhibited kinetic parameters for DCIP, NADH, and NADPH similar to those of DTD except that, with K76V, the Km for NADPH was doubled. Both mutant proteins contained two molecules of FAD per enzyme molecule. Dicumarol inhibited K76V and C179A mutant activities to greater than 90% at a concentration of 10(-7) M. Heat stability studies showed that C179A was much more sensitive to inactivation at 37 degrees C than both the wild-type and K76V enzymes. It is concluded from this study that lysine 76 and cysteine 179 are not essential in catalysis and in the binding of FAD, DCIP, and dicumarol. However, lysine residue 76 appears to play a role in NADPH binding and cysteine residue 179 is important in maintaining the stability of the enzyme.
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PMID:Site-directed mutagenesis of rat liver NAD(P)H: quinone oxidoreductase: roles of lysine 76 and cysteine 179. 156 99

Rats fed an ethanol-containing diet for 4 weeks showed a 3- to 5-fold increase over isocalorically pair-fed controls with respect to cytosolic NAD(P)H-quinone oxidoreductase (NQOR) (E.C.1.6.99.2) with both menadione and dichlorophenol-indophenol as substrates. Rates of NAD(P)H-dependent p-nitrosophenol (pNSP) reduction catalyzed by rat liver cytosolic fractions were increased 1.5- to 2-fold upon pretreatment of the animal with ethanol. NQOR contributed almost exclusively to the NADPH-dependent C-nitrosoreductase activity in cytosol as judged by the strong inhibition of the reaction by dicoumarol. In contrast, NADH-dependent C-nitrosoreductase activity was inhibited 70-80% by pyrazole and thus may be attributed mainly to alcohol dehydrogenase(s). Highly purified rat liver cytosolic NQOR catalyzed the NADH- and NADPH-dependent reduction of pNSP to p-aminophenol. We therefore suggest that ethanol ingestion enhances the reduction of the C-nitrosoaromatics formed upon cytosolic metabolism of arylamines or nitroarenes by two mechanisms. Increased NADPH-dependent reduction is mediated by the induction of cytosolic NQOR while an NADH-dependent pathway responds to the increased availability of reduced cofactor upon ethanol ingestion and involves mainly the alcohol dehydrogenase-mediated reduction of such compounds.
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PMID:Role of cytosolic NAD(P)H-quinone oxidoreductase and alcohol dehydrogenase in the reduction of p-nitrosophenol following chronic ethanol ingestion. 158 50

The structural gene of the Paracoccus denitrificans NADH-ubiquinone oxidoreductase encoding a homologue of the 75-kDa subunit of bovine complex I (NQO3) has been located and sequenced. It is located approximately 1 kbp downstream of the gene coding for the NADH-binding subunit (NQO1) [Xu, X., Matsuno-Yagi, A., and Yagi, T. (1991) Biochemistry 30, 6422-6428] and is composed of 2019 base pairs and codes for 673 amino acid residues with a calculated molecular weight of 73,159. The M(r) 66,000 polypeptide of the isolated Paracoccus NADH dehydrogenase complex is assigned the NQO3 designation on the basis of N-terminal protein sequence analysis, amino acid analysis, and immuno-cross-reactivity. The encoded protein contains a putative tetranuclear iron-sulfur cluster (probably cluster N4) and possibly a binuclear iron-sulfur cluster. An unidentified reading frame (URF3) which is composed of 396 base pairs and possibly codes for 132 amino acid residues was found between the NQO1 and NQO3 genes. When partial DNA sequencing of the regions downstream of the NQO3 gene was performed, sequences homologous to the mitochondrial ND-1, ND-5, and ND-2 gene products of bovine complex I were found, suggesting that the gene cluster carrying the Paracoccus NADH dehydrogenase complex contains not only structural genes encoding water-soluble subunits but also structural genes encoding hydrophobic subunits.
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PMID:Structural features of the 66-kDa subunit of the energy-transducing NADH-ubiquinone oxidoreductase (NDH-1) of Paracoccus denitrificans. 160 43

A prokaryotic expression plasmid, pKK-DT2, containing the cDNA of rat liver NAD(P)H:quinone-acceptor oxidoreductase (EC 1.6.99.2; DT-diaphorase) was constructed and used to transform Escherichia coli strain JM109. The rat liver quinone reductase was expressed in strain in JM109 and was inducible with isopropyl beta-D-thiogalactopyranoside (IPTG). The expressed rat protein was purified by affinity chromatography and had kinetic and physical properties identical with the protein purified from rat liver in that it could utilize either NADH or NADPH as the electron donor and its activity was inhibited by dicoumarol. In addition, we have generated four mutants, Arg-177----His (R177H), Arg-177----Ala (R177A), Arg-177----Cys (R177C) and Arg-177----Leu (R177L), using this expression system. Several of the mutants behaved anomalously on SDS/PAGE, but all of the mutant proteins had the expected M(r) as determined by electrospray m.s. These results and those obtained from enzyme kinetic analysis, u.v./visible absorption spectral analysis, and flavin and tryptophan fluorescence analysis of the wild-type enzyme and four mutants indicated that mutations at Arg-177 changed the conformation of the enzyme, resulting in a decrease in enzyme activity. Replacing Arg-177 with leucine altered the protein conformation and decreased FAD incorporation.
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PMID:Expression of rat liver NAD(P)H:quinone-acceptor oxidoreductase in Escherichia coli and mutagenesis in vitro at Arg-177. 162 1

Eleven frostbites were induced on the ears of seven New Zealand White rabbits and specimens were taken from the lesion after 1, 4 and 8 hours, and from ten further frostbites on the ears of six rabbits for examination 1, 3 and 7 days later. The specimens were taken at the border between the frozen and non-frozen skin. NADH-diaphorase, alkaline phosphatase and esterase were demonstrated histochemically in the sample, which was also studied by haematoxylin and eosin staining. Five ears served as controls. Some granulocytes could be seen accumulating in the vessels and in the dermis at the border of the frostbite area after only 1 hour, and other enzyme rich cells (macrophages) also began to appear. After 4 hours the inflammation was quite obvious with the enzyme reactions clearly observable in the sections. After 8 hours there was no marked difference compared with the 4-hour picture. It was only after 3 days that the line of demarcation between the normal and frostbite tissue could be seen clearly. This was oblique in some specimens and vertical in others. The degeneration in the lesion could best be demonstrated by the NADH-diaphorase and esterase reactions and the early inflammation by the alkaline phosphatase reaction.
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PMID:Enzyme histochemical reactions at the demarcation line in frostbite: an experimental study on rabbits. 162 43

Bacterial respiratory chain has two types of NADH-quinone reductase (NQR): one is energy-coupled (type-1) and the other had no energy-transducing capacity, that is, energy-uncoupled (type-2). Each of the NADH-reacting flavoprotein subunits of NQR-1 from Escherichia coli and the marine Vibrio alginolyticus reduced quinone to semiquinone radicals by the one-electron transfer pathway and was very sensitive to preincubation with NADH. On the other hand, the NQR-2 from these bacteria reduced quinone to quinol by the two-electron transfer pathway and was insensitive to preincubation with NADH. Since the NQR-1 from E. coli functions as a proton pump, whereas that from the marine V. alginolyticus functions as a sodium pump, the formation of semiquinone radicals as an intermediate is likely to be a common mechanism to functioning as either proton or sodium pump.
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PMID:Characteristic differences in the mode of quinone reduction and stability between energy-coupled and -uncoupled NADH-quinone reductases from bacterial respiratory chain. 162 43

Formation of the nasal septal cartilage in prenatal and neonatal rats was studied histologically and by histochemistry to determine the manner, degree and participation of the nasal septal cartilage in midface growth and in bone formation of the face. Chondrogenesis of the nasal septal cartilage started at the 13th embryonic day, premaxillary and vomerin bone formation at the 14th embryonic day and endochondral bone formation of the septo-presphenoid area at the 17th embryonic day. After differentiation of the nasal septal cartilage, this cartilage supported ethmoid bone formation by endochondal ossification in the septo-presphenoid area. Nasal septal cartilage showed intense activity of lactate dehydrogenase, NADH2-diaphorase and a moderate activity of acid phosphatase, whereas premaxillary and vomerin bone showed intense activity of alkaline phosphatase. Osteoblasts showed intense activity of alkaline phosphatase, lactate dehydrogenase and NADH2-diaphorase and osteoclasts showed intense activity of acid phosphatase. During the embryonal period growth of the nasal septal cartilage could occur in an ethmoido-rostral direction supported by endochondral ossification and growth in length and height supported by apposition and interstitial growth.
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PMID:Histochemical analysis of enzymes involved in the formation and metabolism of the nasal septal cartilage. 163 42

Human or rat microsomal 5 alpha-reductase activity, as measured by enzymic conversion of testosterone into 5 alpha-dihydrotestosterone or by binding of a competitive inhibitor, [3H]17 beta-NN-diethulcarbamoyl-4-methyl-4-aza-5 alpha-androstan-3-one ([3H]4-MA) to the reductase, is inhibited by low concentrations (less than 10 microM) of certain polyunsaturated fatty acids. The relative inhibitory potencies of unsaturated fatty acids are, in decreasing order: gamma-linolenic acid greater than cis-4,7,10,13,16,19-docosahexaenoic acid = cis-6,9,12,15-octatetraenoic acid = arachidonic acid = alpha-linolenic acid greater than linoleic acid greater than palmitoleic acid greater than oleic acid greater than myristoleic acid. Other unsaturated fatty acids such as undecylenic acid, erucic acid and nervonic acid, are inactive. The methyl esters and alcohol analogues of these compounds, glycerols, phospholipids, saturated fatty acids, retinoids and carotenes were inactive even at 0.2 mM. The results of the binding assay and the enzymic assay correlated well except for elaidic acid and linolelaidic acid, the trans isomers of oleic acid and linoleic acid respectively, which were much less active than their cis isomers in the binding assay but were as potent in the enzymic assay. gamma-Linolenic acid had no effect on the activities of two other rat liver microsomal enzymes: NADH:menadione reductase and glucuronosyl transferase. gamma-Linolenic acid, the most potent inhibitor tested, decreased the Vmax. and increased Km values of substrates, NADPH and testosterone, and promoted dissociation of [3H]4-MA from the microsomal reductase. gamma-Linolenic acid, but not the corresponding saturated fatty acid (stearic acid), inhibited the 5 alpha-reductase activity, but not the 17 beta-dehydrogenase activity, of human prostate cancer cells in culture. These results suggest that unsaturated fatty acids may play an important role in regulating androgen action in target cells.
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PMID:Inhibition of steroid 5 alpha-reductase by specific aliphatic unsaturated fatty acids. 163 46

This study investigates the cytotoxic and genotoxic effects of various carboxy AQ, 1,4-dihydroxy 6-carboxy AQ, 1,8-dihydroxy 3-carboxy AQ, 1,4-dihydroxy AQ, 1,5-dihydroxy AQ, 1,8-dihydroxy AQ and 2,6-dihydroxy AQ in V79 Chinese hamster cells. The V79 cells were used since, as they contain flavoproteins but not cytochrome P-450, they can bioactive xenobiotics only through the reductive pathway excluding the oxidative one. In addition, the abilities of AQs to stimulate O2-production using both purified flavoproteins (NADH-dehydrogenase, NADPH-cytochrome P-450 reductase) and V79 subcellular fractions (homogenate and microsomes) were assayed. The NADH and NADPH consumption stimulated by AQs in V79 microsomes was also determined. The results showed that the carboxylic-containing drugs and the 1,4-dihydroxy AQ were weak sister chromatid exchange inducers and the most toxic among the six anthraquinones examined. Dicumarol, a potent inhibitor of DT-diaphorase, reduced, rather than potentiated, both the cytotoxicity and genotoxicity caused by these AQs. Thus, the higher superoxide formation rates stimulated by the carboxylic-containing AQs compared to those of the other quinones with all the in vitro systems used, suggested, except for the 1,4-dihydroxy AQ, a possible relationship between cytotoxicity and O2-production. For the 1,4-dihydroxy AQ toxicity, a specific bioactivation route was hypothesized.
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PMID:Superoxide anion production and toxicity in V79 cells of six hydroxy-anthraquinones. 165 52

1. Mixed-function oxidase (MFO) system components (cytochrome P-450, "418-peak", cytochrome b5 and NADPH-cytochrome c(P-450) reductase) and inducible antioxidant enzymes (catalase, superoxide dismutase (SOD), glutathione peroxidase (GPX) and DT-diaphorase) has been determined in digestive glands of mussels (Mytilus galloprovincialis) collected from three Mediterranean coastal locations, exhibiting an organic pollution gradient. 2. Cytochrome P-450, the "418-peak", catalase and SOD showed a good correlation with whole body tissue PAHs and, to a lower extent, with PCBs. 3. Microsomal NADPH-dependent DT-diaphorase, but not the NADH-dependent microsomal enzyme or the cytosolic DT-diaphorases, was indicated to increase with pollution exposure. 4. The application of such measurements to environmental monitoring is discussed. Given the magnitude of differences observed, and the state of knowledge on enzyme function and mechanisms of toxicity, a multiparameter approach is considered to offer current and future potential for detecting the impact of organic pollution on bivalve molluscs.
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PMID:Responses of mixed-function oxygenase and antioxidase enzyme system of Mytilus sp. to organic pollution. 167 52


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