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)

NAD(P)H: quinone oxidoreductase (NQO1) is believed to be protective against cancer and toxicity caused by exposure to quinones and their metabolic precursors. This enzyme catalyzes the two-electron reduction of compounds, compared with one-electron reduction mediated by NADPH: cytochrome-P450 oxidoreductase which produces toxic and mutagenic free radicals. Recently we cloned and sequenced the cDNA encoding human 2.3,7,8-tetrachlorodibenzo-p-dioxin (dioxin)-inducible cytosolic NQO1 [Jaiswal et al. (1988) J. Biol. Chem. 263, 13572-13578] and provided preliminary evidence that this enzyme may correspond to diaphorase 4, an enzymatic activity present in various tissues that catalyzes the reduction of a variety of quinones by both NADH and NADPH [Edwards et al. (1980) Biochem. J. 187, 429-436]. In the present report we characterize the catalytic properties of the protein encoded by the NQO1 cDNA. The enzyme was synthesized in monkey kidney COS-1 cells transfected with a pMT2-based expression plasmid containing the NQO1 cDNA. Western blot analysis of the transfected cells using an antibody against rat liver cytosolic NQO1 revealed a 31-kDa band that was not detected in nontransfected cells. This band corresponded to a polypeptide with the same electrophoretic mobility as the endogenous NQO1 protein detected in the human hepatoblastoma (Hep-G2) cells with the same antibody. The immunoreactive protein detected in human Hep-G2 cells was induced approximately fourfold by exposure of the cultures to dioxin, an increase commensurate with the increased in quinone oxidoreductase activity. These studies suggest that the protein encoded by NQO1 cDNA is indeed similar, if not identical, to the dioxin-inducible protein band detected in human Hep-G2 cells. Further characterization of the product of NQO1 cDNA, which was present at approximately 20-30-fold higher levels in transfected COS cells than the endogenous product in uninduced human Hep-G2 cells indicated that it had very high capacity (greater than 1000-fold over background) to catalyze the reduction of 2.6-dichloroindophenol and menadione. Besides these two commonly used substrates for quinone reductase, the expressed NQO1 protein also effectively metabolized 2,6-dimethylbenzoquinone, methylene blue, p-benzoquinone, 1,4-naphthoquinone, 2-methyl-1,4-benzoquinone, with the latter being the most potent electron acceptor at 50 microM concentration of the substrate.
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PMID:The human dioxin-inducible NAD(P)H: quinone oxidoreductase cDNA-encoded protein expressed in COS-1 cells is identical to diaphorase 4. 189 80

We have previously reported the isolation of CHO cell lines resistant to mitomycin C under aerobic conditions of drug exposure. Here it is reported that these cell lines have the same response to mitomycin C under hypoxic conditions as do controls. The cells are shown to have lower levels of DT-diaphorase activity than controls, but similar levels of activity of NADPH:cytochrome c reductase, another enzyme involved in the metabolism of mitomycin C. Evidence for molecular defects in the DT-diaphorase gene or gene transcript is presented for the deficient cell lines. The consequences of this DT-diaphorase deficiency is further explored by testing the toxicity of menadione, an established enzyme substrate. The isolation of CHO cell lines deficient in DT-diaphorase activity and resistant to mitomycin C under aerobic but not hypoxic conditions suggests that mitomycin C reduction by this enzyme has a significant impact on cytotoxicity under aerobic but not hypoxic conditions. Similarly, DT-diaphorase metabolism of menadione does not appear to have a significant impact on cytotoxicity in CHO cells.
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PMID:Chinese hamster ovary cell lines resistant to mitomycin C under aerobic but not hypoxic conditions are deficient in DT-diaphorase. 190 Jul 39

The activity of ferredoxin: NADP+ reductase (FNR) was found to decline to approximately 20% maximal levels with little or no loss in enzyme levels when cultures of the cyanobacterium Anabaena variabilis were maintained in the stationary phase of growth. Re-activation of enzyme activity occurred when cells were diluted into either fresh or re-utilized media and illuminated. This reversible de-activation/re-activation process was found, in vivo, to be dependent on the intensity of light illuminating the cells. The de-activated form of FNR was purified to homogeneity and exhibited the same molecular mass, isoelectric-focusing pattern and N-terminal amino acid sequence as the native form. Both de-activated and native FNR preparations each exhibited three reactive thiol groups on denaturation in urea; however, the rate of reaction with Ellman's reagent was much faster with the de-activated form than with the native form. Both preparations contain a single disulphide bond. Upon reduction of the disulphide bond in either form of the enzyme, the five reactive thiol groups exhibited identical reactivities in the presence of urea. Steady-state kinetic analysis of the de-activated form showed a marked increase in Km values for NADPH in diaphorase assays and an increase in Km for ferredoxin in the ferredoxin-mediated reduction of cytochrome c. No significant difference in kcat. was observed in comparison of the de-activated with the native form in any of the above assays; however, the de-activated form did exhibit a lower kcat. value in the transhydrogenase assay. The de-activated form of FNR bound ferredoxin with a 16-fold lower affinity than the native enzyme. These data suggest that the de-activation of FNR in vivo in response to low light intensity involves an alteration in protein structure, possibly via an intramolecular thiol disulphide interchange, which influences the interaction of the enzyme with its substrates.
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PMID:Light-dependent de-activation/re-activation of Anabaena variabilis ferredoxin: NADP+ reductase. 190 89

Dinitropyrenes (DNP) are potent bacterial mutagens in the Ames test and genotoxins in cultured mammalian cells. Rat liver cytosol contains nitroreductases that are critical in the activation of DNP to the ultimate DNA-binding species. In order to study the nature and inducibility of liver cytosolic enzymes involved in the activation of DNP, cytosolic nitroreductase activities towards three DNP isomers (1,3-, 1.6- and 1,8-DNP) were determined in Aroclor-pretreated and untreated rats. Aroclor-1254 pretreatment resulted in up to 5-fold induction of cytosolic DNP nitroreductase. This induction was reflected in at least a 15-fold increase in cytosolic NAD(P)H-quinone oxidoreductase (NQOR) (E.C. 1.6.99.2) activity. The rates of nitroreduction for the three DNP isomers followed the order 1,6- greater than 1,8- greater than 1,3-DNP in all cases studied. 1,6-DNP nitroreductase coeluted with NQOR activity upon affinity purification. Highly purified NQOR catalyzed the NADH- and NADPH-dependent reduction of each of the three DNP isomers and displayed the same stereospecificity as the cytosolic activity. These results provide evidence that NQOR participates in the cytosolic nitroreduction of DNP and constitutes a major part of the total DNP nitroreductase activity upon induction of NQOR by Aroclor-1254 pretreatment. Thus, the role of NQOR in the metabolism of these mutagens depends significantly upon the degree to which this enzyme is induced.
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PMID:Dinitropyrene nitroreductase activity of purified NAD(P)H-quinone oxidoreductase: role in rat liver cytosol and induction by Aroclor-1254 pretreatment. 190 25

Pyruvate:NADP+ oxidoreductase from Euglena gracilis, a homodimeric protein with a molecular weight of 309 kDa, is an iron-sulfur flavoenzyme that contains thiamin pyrophosphate (TPP). The functional structure of the enzyme was studied by a limited proteolysis experiment using trypsin. The evidence obtained shows that the enzyme consists of two functional domains, one of which contains an iron-sulfur cluster, which can be isolated as a homodimeric fragment of approximately 220 kDa by proteolysis. The other domain that contains FAD is released as a monomeric fragment of approximately 55 kDa. The pyruvate dehydrogenase reaction is still catalyzed by the large fragment when NADP+ is substituted by methyl viologen, while the small fragment retains a diaphorase-like electron-transfer activity from NADPH to MV. It is thus shown that pyruvate is oxidized in a CoA-dependent reaction to form CO2 and acetyl-CoA in the iron-sulfur domain, and that the two electrons formed are transferred to the FAD domain in which NADP+ is reduced. TPP is considered to be associated in the iron-sulfur domain. The NH2-terminal sequences of the enzyme and its proteolytic fragments reveal that the iron-sulfur domain occurs in the NH2-terminal side of the enzyme. For elucidation of the O2 instability of the enzyme, limited proteolysis was attempted in air. The tryptic fragment derived from the iron-sulfur domain, similar to the native enzyme, appears to be inactivated by direct contact with O2. In contrast, the FAD domain, when separated from the other domain, is quite stable in air, although the diaphorase activity decays when the native enzyme is exposed to O2.
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PMID:Pyruvate:NADP+ oxidoreductase from Euglena gracilis: limited proteolysis of the enzyme with trypsin. 191 Feb 87

Carbonyl reductase (NADPH: secondary-alcohol oxidoreductase; EC 1.1.1.184), a widely distributed NADPH-dependent enzyme considered as both an aldo-keto reductase and a quinone reductase, was cloned from a human liver genomic library and transiently expressed in COS7 cells. The gene contains 3142 bases comprising three exons and two introns. The absence of a CAAT and TATA box and the presence of a GC-rich island are characteristic of many "housekeeping" genes. Transient expression of the genomic gene in COS7 cells using an expression vector containing an SV40 origin of replication resulted in a greater than 50-fold increase in both menadione reductase activity and daunorubicin reductase activity, suggesting that both activities are derived from the same enzyme. Carbonyl reductase mRNA levels reflected enzyme activity levels in the transfected cells. Other parameters, such as pH profile, cofactor requirements, substrates, and inhibitors, were similar to those of carbonyl reductase purified by other investigators. Potential regulatory elements with consensus sequences for two GC boxes and the transcriptional activator protein AP-2 were present upstream of the transcriptional start site. Although the precise role of carbonyl reductase is unknown, the enzyme is involved in drug metabolism and in the reduction of activated carbonyl compounds. Its ability to act as a quinone reductase also implies a potential to modulate oxygen free radicals.
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PMID:Genomic sequence and expression of a cloned human carbonyl reductase gene with daunorubicin reductase activity. 192 84

Quinones can be metabolized by various routes: substitution or reductive addition with nucleophilic compounds (mainly glutathione and protein thiol groups), one-electron reduction (mainly by NADPH: cytochrome P-450 reductase) and two-electron reduction (by D,T-diaphorase). During reduction semiquinone radicals and hydroquinones are formed, which can transfer electrons to molecular oxygen, resulting in the formation of reactive oxygen intermediates and back-formation of the parent quinone (redox cycling). Reaction of semiquinones and reactive oxygen intermediates with DNA and other macromolecules can lead to acute cytotoxicity and/or to mutagenicity and carcinogenicity. The enhanced DNA-alkylating properties of certain hydroquinones are exploited in the bioreductive alkylating quinones. Acute cytotoxicity of quinones appears to be related to glutathione depletion and to interaction with mitochondria and subsequent disturbance of cellular energy homoeostasis and calcium homoeostasis. These effects can to a certain extent be predicted from the electron-withdrawing and electron-donating effects of the substituents on the quinone nucleus of the molecule. Prediction of cytostatic potential remains much more complicated, because reduction of the quinones and the reactivity of the reduction products with DNA are modulated by the prevailing oxygen tension and by the prevalence of reducing enzymes in tumour cells.
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PMID:Bioreductive activation of quinones: a mixed blessing. 192 1

Injection of Fast Blue into different cortical areas (frontal, parietal, anterior and posterior cingulate cortex) revealed that neurons in the white matter (interstitial neurons) give rise to association fibers which project mostly to the gray matter of the overlying cytoarchitectonic area, but which may extend also over different cytoarchitectonic areas. The rostrocaudal extent of the projecting axons was up to 1 mm in the frontal and parietal cortex, and up to 3.5 mm in the cingulate cortex. Concurrent processing for dihydronicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) histochemistry showed that 70% of cortically projecting interstitial neurons were NADPH-d-positive. An analysis of neuronal morphology suggests that the FasT-Blue-labeled, NADPH-d-negative neurons may represent displaced pyramidal neurons of layer VIb; the Fast-Blue-labeled and NADPH-d-positive neurons have bipolar or multipolar dendritic trees, constituting a population of nonpyramidal interstitial neurons that project into the cortical gray matter.
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PMID:The efferent projections of neurons in the white matter of different cortical areas of the adult rat. 192 49

The diaphorase activity of NADPH: adrenodoxin reductase (EC 1.18.1.2) is stimulated by adrenodoxin. The latter prevents the reductase inhibition by NADPH; the Line-weaver-Burk plots are characterized by a biphasic dependence of the reaction rate on the oxidizer concentration. At pH 7.0 the maximal rate of the first phase is 20s-1; that for the second phase at saturating concentrations of adrenodoxin is 5 s-1. Since the second phase rate is equal to that of the adrenodoxin-linked cytochrome c reduction by reductase it is concluded that this phase reflects the reduction of the oxidizers via reduced adrenodoxin. Quinones are reduced by adrenodoxin in an one-electron way; the logarithms of their rate constants depend hyperbolically on their single-electron reduction potentials (E7(1]. The oxidizers interact with a negatively charged domain of adrenodoxin. The depth of the adrenodoxin active center calculated from the Fe(EDTA)- reduction data is 5.9 A.
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PMID:[Stimulation of the NADPH:adrenodoxin reductase diaphorase reaction by adrenodoxin]. 207 39

A flavoprotein with properties similar to those of ferredoxin:NADP+ oxidoreductases found in the leaves of higher plants has been purified to apparent homogeneity from bean sprouts, a nonphotosynthetic plant tissue. The absorbance and circular dichroism spectra of the bean sprout protein are similar to those of spinach leaf ferredoxin:NADP+ oxidoreductase and an antibody raised against the spinach enzyme recognized the bean sprout enzyme. The bean sprout enzyme catalyzed ferredoxin-dependent electron transfer from NADPH to equine cytochrome c at a high rate but, unlike the spinach enzyme, exhibited little NADPH to 2,6-dichlorophenol indophenol diaphorase activity. The bean sprout enzyme forms a 1:1 electrostatically stabilized complex with ferredoxins isolated from either bean sprouts or spinach leaves.
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PMID:Characterization of a ferredoxin:NADP+ oxidoreductase from a nonphotosynthetic plant tissue. 210 79


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