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)

The cytochrome b subunit of the ubiquinol:cytochrome c oxidoreductase (the bc1 complex) contains two heme prosthetic groups, cytochrome bL and cytochrome bH. In addition, this subunit also provides major elements of the quinol oxidation site (Qo) and a separate quinone reductase site (Qi), which are thought to be located on opposite sides of the membrane. Site-directed mutagenesis has been used to explore the role(s) of specific amino acid residues in this subunit from the photosynthetic bacterium Rhodobacter sphaeroides. Previous work identified five residues, Gly48 (Gly33), Ala52 (Gly37), His217 (His202), Lys251 (Lys228), and Asp252 (Asp229), as being either at or near the quinone reductase site (the residue numbers in parentheses designate the equivalent positions in the yeast mitochondrial enzyme). These residues are predicted to be near the cytoplasmic boundaries of transmembrane helices: helix A (G48, A52), helix D (H217), or helix E (K251, D252). In the current work, the importance of two additional highly conserved residues, which are also predicted to be near the cytoplasmic boundaries of transmembrane helices, is explored by site-directed mutagenesis. R114 (helix B) has been substituted with K, Q, and A, and W129 (helix C) has been changed to A and F. The results suggest that a positively charged residue at position 114 is important. The R114K mutation causes only subtle effects, which appear to be localized to cytochrome bH and the quinone reductase site. In contrast, R114Q is not assembled, and R114A, although partially assembled, is nonfunctional and appears to have a very low amount of cytochrome b associated with the complex. Both mutants at position 129 (W129A and W129F) are able to support the photosynthetic growth of the organism, but show abnormal characteristics. The defects associated with the W129A mutation appear to be primarily associated with the quinone reductase site and cytochrome bH, whereas the W129F mutation appears to result in more global defects that also perturb the cytochrome bL locus. The results are consistent with the placement of residues R114 and W129 near the cytoplasmic side of the membrane, but suggest that these residues are important for the assembly and overall stability of the complex.
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PMID:Site-directed mutagenesis of arginine-114 and tryptophan-129 in the cytochrome b subunit of the bc1 complex of Rhodobacter sphaeroides: two highly conserved residues predicted to be near the cytoplasmic surface of putative transmembrane helices B and C. 794 7

An antibody to cytochrome P450 oxidoreductase, purified from rat liver, has been used for the immunohistochemical localization of cytochrome P450 oxidoreductase-like immunoreactivity in the rat central nervous system. The distribution of this immunoreactivity has been confirmed using in situ hybridization with specific cytochrome P450 oxidoreductase antisense DNA probes. Cytochrome P450 oxidoreductase immunoreactivity was detected in neurons and was found in some glial populations. Immunoreactivity and in situ messenger RNA signals were present in many forebrain areas including the olfactory bulb, in the cerebral cortex, caudate-putamen, globus pallidus, hypothalamus, thalamus and hippocampus. Cytochrome P450 oxidoreductase was also detected in the nucleus of the posterior commissure, superior colliculus, intermediate gray layer, periaqueductal gray and in the molecular, Purkinje and granular layers of the cerebellum. In the brain stem, cytochrome P450 oxidoreductase was detected in the substantia nigra, nucleus locus coeruleus and raphe nucleus. Western blotting studies revealed the brain immunoreactive protein has a mol. wt of approximately 72,000, as reported for cytochrome P450 oxidoreductase purified from rat brain microsomes. The distribution of cytochrome P450 oxidoreductase immunoreactivity was compared with the distribution of cells exhibiting NADPH diaphorase activity, which has been established as a histochemical marker for neuronal nitric oxide synthase, an enzyme which has a C-terminus with some structural similarity with cytochrome P450 oxidoreductase and catalyses a complex reaction resulting in the synthesis of nitric oxide from arginine. In general, cytochrome P450 oxidoreductase immunoreactivity and nitric oxide synthase diaphorase activity did not co-localize; however, some neuronal populations did express nitric oxide synthase and exhibit cytochrome P450 oxidoreductase immunoreactivity. Results of immunohistochemistry and in situ hybridization experiments suggest cytochrome P450 oxidoreductase is widespread in the rat central nervous system. The distribution pattern of cytochrome P450 oxidoreductase did not match with those of any one neurotransmitter; however, it did coincide with some brain regions known to harbour central catecholaminergic neurons. The general distribution of cytochrome P450 oxidoreductase was similar to the distribution reported for haeme oxygenase 2 and several cytochrome P450 enzymes. It is possible that malfunctions in cytochrome P450 enzyme systems and/or the haeme oxygenase 2 pathways, both of which involve cytochrome P450 oxidoreductase, may have implications in neurodegenerative diseases.
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PMID:Localization of NADPH cytochrome P450 oxidoreductase in rat brain by immunohistochemistry and in situ hybridization and a comparison with the distribution of neuronal NADPH-diaphorase staining. 796 13

NAD(P)H:quinone acceptor oxidoreductase (NQO1, EC 1.6.99.2) is an enzyme that is believed to play a central role in the bioreductive activation of several compounds, particularly quinones. The results of this study demonstrate that the activity of NQO1 is significantly elevated (2.5-fold) in HT-29 human colon cells that are in the plateau phase of the growth curve as opposed to cells in the exponential phase. Analysis of gene expression using semiquantitative reverse transcription-polymerase chain reaction and Northern blot analysis demonstrates that the increased enzyme activity is associated with increased NQO1 mRNA levels. Sequential trypsinization of layers of cells from HT-29 multicellular spheroids and analysis of gene expression by reverse transcription-polymerase chain reaction demonstrate that NQO1 expression is elevated in cells close to the necrotic center. Maximum expression occurs at a depth of 90-110 microns, with reduced expression as the distance toward both the surface and the necrotic center decreases. HT-29 spheroids were significantly more responsive than monolayers (concentration producing 50% inhibition, 124.6 and 364 nM, respectively) to the experimental drug, 2,5-dimethyl-3,6 diaziridinyl-1,4 benzoquinone. While the environmental stimulus responsible for causing elevated NQO1 expression has not been identified, the fact that NQO1 expression is influenced by microenvironmental conditions will have important implications for those drugs that are activated by NQO1.
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PMID:Increased activity and expression of NAD(P)H:quinone acceptor oxidoreductase in confluent cell cultures and within multicellular spheroids. 803 96

The NAD(P)H-flavin oxidoreductase gene from the bioluminescent bacterium, Vibrio fischeri ATCC 7744, was expressed in Escherichia coli, and the enzyme purified using Cibacron Blue 3G-A affinity column chromatography from crude extracts in a single step. The purified enzyme had a typical flavoprotein absorption spectrum and flavin mononucleotide (FMN) was identified as a prosthetic group, non-covalently bound in a molar ratio of 1:1. The enzyme catalyzed the electron transfer from NADH via FMNH2 to various other electron acceptors. Reduced flavin produced by flavin reductase participated non-enzymatically in the following reactions: H2O2-forming NADH oxidase-like, oxygen-insensitive nitroreductase-like, diaphorase (quinone reductase)-like and bacterial luciferase reactions.
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PMID:NAD(P)H-flavin oxidoreductase from the bioluminescent bacterium, Vibrio fischeri ATCC 7744, is a flavoprotein. 803 96

Many solid tumors contain substantial fractions of hypoxic cells which are relatively resistant to both radiation therapy and certain cytotoxic drugs. We have previously shown that exposure of human HT29 cells to hypoxic conditions results in the overexpression of certain enzymes involved in the detoxication of xenobiotics, including NAD(P)H:(quinone acceptor) oxidoreductase (DT)-diaphorase, and gamma-glutamylcysteine synthetase, the rate-limiting enzyme in glutathione synthesis. This hypoxic effect on DT-diaphorase was shown to involve both transcriptional induction and altered message stability. We have investigated the effects of hypoxia on elements in the promoter region of DT-diaphorase. Electrophoretic mobility shift assays demonstrate the induction of a binding activity to the AP-1 response element of DT-diaphorase. Supershift assays suggest that this binding is due to AP-1 nuclear factors and that members of the jun family are induced to a greater degree than fos by hypoxia. Analysis of the kinetics of transcription factor expression indicates that the expression of c-jun and junD is induced during hypoxic exposure; mRNA levels fall during reoxygenation. Induction of fos on the other hand is not as florid during hypoxia (5-fold) and is most pronounced (17-fold) 24 h after the restoration of an oxic environment. Thus, the hypoxic response of DT-diaphorase expression is mediated in part through AP-1, initially by a jun-related mechanism and then by the involvement of fos. The affinity of transcription factors for the AP-1 binding site depends on the redox state of a cysteine residue located close to the DNA-binding region of both Fos and Jun. A nuclear protein, Ref-1, maintains the reduced state of Fos and Jun and promotes binding to AP-1. Nuclear extracts of HT29 cells exposed to hypoxia show markedly increased Ref-1 protein content. Elevation of ref-1 steady-state mRNA levels occurs as an early event following induction of hypoxia and persists when cells are restored to a normally oxygenated environment. Nuclear run-on analysis demonstrates that induction of transcription is the mechanism of ref-1 mRNA elevation. Electrophoretic mobility shift assays and immunodepletion assays were used to further define the interaction of Ref-1 with specific AP-1-binding proteins under hypoxic conditions. These data demonstrate that the induction of detoxicating enzyme expression in HT29 cells exposed to hypoxia results from the induction of both transactivating factors that bind to the AP-1 element and of redox proteins that enhance their affinity for this element.
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PMID:Activation of AP-1 and of a nuclear redox factor, Ref-1, in the response of HT29 colon cancer cells to hypoxia. 806 32

Three related Chinese hamster ovary (CHO) cell lines derived from CHO-K1R cells (MMC3-A2, 21-1 and G1B) previously shown to differ in their sensitivity to mitomycin C (MMC), were investigated in more detail to determine the factors controlling this sensitivity. A separately maintained wild type cell line (CHO-K1TOR) was included in this study for comparison. Continuous (chronic) exposure of the five cell lines to MMC during the 10-day colony forming assay demonstrated a 15-fold range in MMC sensitivity between the most sensitive cell line (MMC3-A2) and the most resistant cell line (G1B) with CHO-K1R, 21-1 and CHO-K1TOR falling at intermediate levels. Acute aerobic exposure (0-5 h) to MMC resulted in a reduced fivefold range of sensitivities, which was further reduced to a three-fold range under hypoxic exposure conditions. These results were suggestive of differences in the aerobic enzymatic activation of MMC as a possible mechanism contributing to the varying sensitivities. There was no correlation between the one-electron reducing enzyme NADPH:cytochrome P-450 oxidoreductase (P450R) activity and cellular sensitivity to MMC. The five cell lines had similar levels of reduced glutathione (GSH), suggesting that oxygen homeostasis was not correlated with the cells, differing sensitivity to MMC. A correlation did exist between NAD(P)H:quinone oxidoreductase (DT-diaphorase) activity and cellular sensitivity to MMC under chronic exposure conditions for the cell lines. High DT-diaphorase levels were also correlated with a reduced ability of oxygen to modulate MMC toxicity. Levels of P450R and DT-diaphorase were not altered significantly during five-hour aerobic or hypoxic exposures of control cells.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Characterization of a set of Chinese hamster ovary variant cell lines demonstrating differing sensitivity to mitomycin C. 812 41

The product formed from 4-nitroquinoline 1-oxide (4NQO), a potent carcinogen, by the action of mouse NADH:4NQO nitroreductase NR-1 was directly identified as 4-hydroxyaminoquinoline 1-oxide (4HAQO) by high performance liquid chromatography analyses in two systems. In liver cytosols from both male and female mice, NADH:4NQO nitroreductase was the predominant enzyme catalyzing the reduction of 4NQO. Rat liver cytosol catalyzed the conversion of 4NQO to either 4HAQO or a glutathione conjugate depending upon coenzyme or cosubstrate availability. Whereas NAD(P)H:quinone reductase (NAD(P)H:(quinone acceptor) oxidoreductase; DT diaphorase; EC 1.6.99.2) was the predominant 4NQO reductase present in liver cytosol from Sprague-Dawley rats, dicumarol-resistant NADH:4NQO nitroreductase specific activities were comparable with those of mouse liver cytosols. A 4NQO nitroreductase from rat liver cytosol was separated from NAD(P)H:quinone reductase chromatographically and shown to have a strong preference for NADH and to be insensitive to inhibition by dicumarol.
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PMID:Conversion of 4-nitroquinoline 1-oxide (4NQO) to 4-hydroxyaminoquinoline 1-oxide by a dicumarol-resistant hepatic 4NQO nitroreductase in rats and mice. 821 72

The levels of NAD(P)H:(quinone-acceptor) oxidoreductase (EC.1.6.99.2) (DT-diaphorase) mRNA and enzyme activity have been studied in paired human normal lung and non-small cell lung tumor samples from patients with a history of cigarette smoking. There were significantly higher levels of DT-diaphorase mRNA (1.2 kilobases) in lung tumor compared to normal lung tissue of patients who had stopped smoking more than 6 months before surgery, with relative values (normalized to beta-actin mRNA) of 29.6 +/- 7.8 (SE) in the lung tumor compared to 11.7 +/- 2.2 in normal lung tissue (P < 0.05). There was no significant difference in DT-diaphorase mRNA between lung tumor and normal lung tissue of subjects who were smokers at the time of surgery, with values of 16.5 +/- 2.1 and 15.3 +/- 2.5 (P > 0.05), respectively. DT-diaphorase enzyme activity in normal and tumor lung tissue was positively correlated with DT-diaphorase mRNA (r = 0.908, P < 0.01). The results of the study suggest that DT-diaphorase does not function as an inducible protectant enzyme in human lung against oxidant species and carcinogens present in cigarette smoke. Metabolism of some anticancer drugs by DT-diaphorase can alter their activity. Differences in DT-diaphorase between lung tumors of smokers and past smokers might alter the response to these drugs.
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PMID:Cigarette smoking is a determinant of DT-diaphorase gene expression in human non-small cell lung carcinoma. 822 86

Established cell lines derived from newborn livers of c14CoS/c14CoS and cch/cch mice have been shown to be genetically resistant (14CoS/14CoS cells) or susceptible (ch/ch cells) to menadione toxicity. These differences are due in part to relatively higher levels of reduced glutathione (GSH) and NAD(P)H:menadione oxidoreductase (NMO1) activity in the 14CoS/14CoS cells. The indolic membrane-stabilizing antioxidant 5,10-dihydroindeno[1,2-b]indole (DHII) was shown previously to protect against various hepatotoxicants in vivo and in primary rat hepatocytes. This report describes how the 14CoS/14CoS and ch/ch cell lines provide a valuable experimental system to distinguish the mechanism of chemoprotection by DHII from menadione toxicity. The addition of 25 microM DHII produced a time-dependent decrease in menadione-mediated cell death in 14CoS/14CoS cells, with little effect on ch/ch cell viability. The maximum protective effect occurred at 24 hr, although the concentration of DHII remained constant for 48 hr. The protective effect of DHII correlated with enhanced glutathione levels (234% increase at 24hr), as well as induction of four enzymes involved in the detoxification and excretion of menadione: NAD(P)H:menadione oxidoreductase (NMO1, quinone reductase), glutathione reductase, glutathione transferase (GST1A1), and UDP glucuronosyltransferase (UGT1*06), with 24-hr maximum induction of 707, 201, 171 and 198%, respectively. Other biotransformation enzymes not directly involved in menadione metabolism (glutathione peroxidase, cytochromes P4501A1 and P4501A2, copper-, zinc-dependent superoxide dismutase, and NADPH cytochrome c oxidoreductase) were not induced by DHII. Menadione-stimulated superoxide production was inhibited 50% by DHII only in 14CoS/14CoS cells, and the inhibition required 24-hr preincubation. Pretreatment with DHII also protected both cell types against the menadione-mediated depletion of GSH, and the increase in percent (oxidized glutathione GSSG), an indicator of oxidative stress. These results suggest that DHII does not protect against menadione toxicity by virtue of its antioxidant or membrane-stabilizing properties. Rather, it acts by inducing a protective enzyme profile that migates redox cycling and facilitates excretion of menadione.
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PMID:Mechanisms of protection from menadione toxicity by 5,10-dihydroindeno[1,2,-b]indole in a sensitive and resistant mouse hepatocyte line. 824 Apr 1

Plasma membranes from most mammalian cells display significant transplasma membrane oxidoreductase (PMO) activity. The enzymes use an extracellular, impermeant electron acceptor as substrate and intracellular reduced pyridine nucleotide as electron donor. The plasma membrane from a neuroblastoma cell line, NB41A3, has been biotinylated and purified by immunoprecipitation with avidin and antiavidin-antibodies. The protein recovery of an immunopurified membrane preparation was < 0.15% of the protein content in the cell extract. The preparation displays an increase in the specific activity of PMO's of 15- to 20-fold compared to the activity in whole cells. With this approach the presence of a NADH-diaphorase within the cell plasma membrane can be demonstrated. This activity accounts for about one third of the total cellular diaphorase activity. The PMO activity cannot be attributed to an increased permeabilization of the plasma membrane induced upon biotinylation nor to intracellular activity from lysed cells. Activation of basal metabolism (glycolysis) stimulates PMO activity up to approx. 54%, presumably through a raise of the intracellular NADH store. PMO also promotes cell growth at low substrate concentrations (0.1-1 microM). Native gel electrophoresis of iminobiotinylated and affinity purified plasma membrane extracts displays two diaphorase-positive bands, indicating that a homogeneous cell population may express several PMO activities at the plasma membrane.
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PMID:An NADH-diaphorase is located at the cell plasma membrane in a mouse neuroblastoma cell line NB41A3. 828


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