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)

EO9 is a novel bioreductive drug which has recently undergone extensive clinical evaluation. Its mechanism of action remains to be clearly defined. Antitumour activity of EO9 has been determined in 2 human colon cancer xenografts (HT-29 and BE) and 2 murine colon adenocarcinomas (MAC 16 and 26) after intratumoural injection of 250 microg of drug. Levels of the major bioreductive enzymes (DT-diaphorase, cytochrome P-450 reductase and cytochrome b5 reductase) were measured in tumours using cytochrome c reduction and menadione as the intermediate electron acceptor. There was no correlation between chemosensitivity (T/C: HT-29, 15%; BE, 27%; MAC 16, 33% and MAC 26, 60%) and enzyme activity (r2 = 0.47 for DT-diaphorase, r2 = 0.1 for cytochrome P-450 reductase and r2 = 0.52 for cytochrome b5 reductase). Drug metabolism was followed in vitro using tumour homogenates incubated under aerobic and anaerobic conditions. Four metabolites were identified by HPLC and characterised bv UV-visible spectroscopy. With the exception of the hydrolysis product EO5A, all other metabolites appeared to be drug adducts. No correlation was observed between the kinetics of metabolite formation and antitumour activity. A good correlation (r2 = 0.86) was found with the rate of disappearance of parent drug and antitumour activity. These data show that the overall capacity of a tumour to metabolise EO9 is the most important determinant of antitumour activity rather than the expression of the major bioreductive enzymes and that the parent drug rather than a metabolite leads to the active form of the drug.
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PMID:Pharmacological and biochemical determinants of the antitumour activity of the indoloquinone EO9. 948 90

The petH genes encoding ferredoxin:NADP+ reductase (FNR) from two Anabaena species (PCC 7119 and ATCC 29413) were cloned and overexpressed in E. coli. Several positively charged residues (Arg, Lys) have been implicated to be involved in ferredoxin binding and electron transfer by cross-linking, chemical modification and protection experiments, and crystallographic studies. The following substitutions were introduced by site-directed mutagenesis: R153Q, K209Q, K212Q, R214Q, K275N, K430Q and K431Q in Anabaena 29413 FNR, and R153E, K209E, K212E, R214E, K275E, R401E, K427E, and K431E in Anabaena 7119 FNR. Comparison of the diaphorase activities, the specific rates of ferredoxin dependent NADP(+)-photoreduction and cytochrome c reduction catalyzed by FNR showed that all these amino acid residues were required for efficient electron transfer between FNR and ferredoxin. Replacement of any one of these basic residues produced a much more pronounced effect on the cytochrome c reductase activity, where FNR, reduced by NADPH, acted as electron donor, than in the reduction of NADP+ by photosystem I via FNR. A mutation involving the replacement of positive charge by a neutral amide produced in all cases a smaller inhibitory effect on the activity than a charge reversal mutation. In addition, it has been found that R214 was necessary for stable integration of the non covalently bound FAD-cofactor.
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PMID:Interaction of positively charged amino acid residues of recombinant, cyanobacterial ferredoxin:NADP+ reductase with ferredoxin probed by site directed mutagenesis. 951 8

1. Mitomycin C (MMC) is considered to be the prototype bioreductive drug undergoing activation to toxic species preferentially under hypoxic conditions. Therefore a proper understanding of the enzymology of bioreduction in tumor tissue is of great importance. 2. DT-diaphorase and NADPH:cytochrome P-450 reductase (quinone reductases) are believed to have established roles in this activation pathway, but these roles are now undergoing revision. 3. It is emerging, however, that different reductases prevail under different physiological conditions. Indeed, DT-diaphorase has been found to protect cells from the hypoxic cytotoxicity of MMC in cell lines expressing high levels of the enzyme. 4. A novel mitochondrial reductase(s) has been identified in solid tumor tissue and is active only under hypoxic conditions and is more efficient at metabolizing MMC than are the other reductases identified. 5. Thus, this newly identified mitochondrial reductase(s) is a potential new target for enzyme-directed bioreductive drug therapy if tumor hypoxia can be achieved. However, because most tumors overexpress DT-diaphorase, this enzyme may prove optimal for MMC drug therapy.
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PMID:Current issues in the enzymology of mitomycin C metabolic activation. 979 12

Humans ingest about 1 g of flavonoids daily in their diet, and they are increasingly being associated with cytoprotective antitumour properties. The mechanism(s) responsible for these effects have not yet been elucidated but may involve interaction with xenobiotic metabolising enzymes to alter the metabolic activation of potential carcinogens. We have investigated the effect of the flavonoids, quercetin (Q), myricetin (M) and epicatechin (E) on the growth, morphology and enzyme activities of MCF7 human breast cancer cells. Of the three flavonoids studied only Q caused a decrease in cell protein content and decreased the reduction of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium). It also inhibited protein, DNA and RNA synthesis to the greatest extent. Q and M increased intracellular reduced glutathione (GSH) content, and Q altered the morphology of the cells after 24 h exposure to 25 microM. E and Q inhibited the O-deethylation of ethoxyresorufin (EROD) catalysed by cytochrome P450 CYPIA. In contrast, M increased the EROD reaction 2-fold. Q increased the activity of DT-diaphorase, NADPH cytochrome c reductase and glutathione reductase, while E increased only NADPH cytochrome c reductase activity. The effects on enzyme activities in vitro suggest that there is not only the potential for flavonoids to alter metabolic activation of carcinogens but also of therapeutically administered drugs in vivo. We are at present investigating the synergy between anti-cancer drugs and flavonoids in terms of anti-tumour efficacy.
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PMID:The effect of the flavonoids, quercetin, myricetin and epicatechin on the growth and enzyme activities of MCF7 human breast cancer cells. 992 Apr 63

Structurally diverse compounds can confer resistance to aflatoxin B1 (AFB1) hepatocarcinogenesis in the rat. Treatment with either phytochemicals [benzyl isothiocyanate, coumarin (CMRN), or indole-3-carbinol] or synthetic antioxidants and other drugs (butylated hydroxyanisole, diethyl maleate, ethoxyquin, beta-naphthoflavone, oltipraz, phenobarbital, or trans-stilbene oxide) has been found to increase hepatic aldo-keto reductase activity toward AFB1-dialdehyde and glutathione S-transferase (GST) activity toward AFB1-8,9-epoxide in both male and female rats. Under the conditions used, the natural benzopyrone CMRN was a major inducer of the AFB1 aldehyde reductase (AFAR) and the aflatoxin-conjugating class-alpha GST A5 subunit in rat liver, causing elevations of between 25- and 35-fold in hepatic levels of these proteins. Induction was not limited to AFAR and GSTA5: treatment with CMRN caused similar increases in the amount of the class-pi GST P1 subunit and NAD(P)H: quinone oxidoreductase in rat liver. Immunohistochemistry demonstrated that the overexpression of AFAR, GSTA5, GSTP1, and NAD(P)H:quinone oxidoreductase affected by CMRN is restricted to the centrilobular (periacinar) zone of the lobule, sometimes extending almost as far as the portal tract. This pattern of induction was also observed with ethoxyquin, oltipraz, and trans-stilbene oxide. By contrast, induction of these proteins by beta-naphthoflavone and diethyl maleate was predominantly periportal. Northern blotting showed that induction of these phase II drug-metabolizing enzymes by CMRN was accompanied by similar increases in the levels of their mRNAs. To assess the biological significance of enzyme induction by dietary CMRN, two intervention studies were performed in which the ability of the benzopyrone to inhibit either AFB1-initiated preneoplastic nodules (at 13 weeks) or AFB1-initiated liver tumors (at 50 weeks) was investigated. Animals pretreated with CMRN for 2 weeks prior to administration of AFB1, and with continued treatment during exposure to the carcinogen for a further 11 weeks, were protected completely from development of hepatic preneoplastic lesions by 13 weeks. In the longer-term dietary intervention, treatment with CMRN before and during exposure to AFB1 for a total of 24 weeks was found to significantly inhibit the number and size of tumors that subsequently developed by 50 weeks. These data suggest that consumption of a CMRN-containing diet provides substantial protection against the initiation of AFB1 hepatocarcinogenesis in the rat.
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PMID:Chemoprevention of aflatoxin B1 hepatocarcinogenesis by coumarin, a natural benzopyrone that is a potent inducer of aflatoxin B1-aldehyde reductase, the glutathione S-transferase A5 and P1 subunits, and NAD(P)H:quinone oxidoreductase in rat liver. 1070 11

In higher plants ferredoxin (Fd):NADP(+) oxidoreductase (FNR) and Fd are each distributed in photosynthetic and non-photosynthetic organs as distinct isoproteins. We have cloned cDNAs for leaf FNR (L-FNR I and L-FNR II) and root FNR (R-FNR) from maize (Zea mays L.), and produced recombinant L-FNR I and R-FNR to study their enzymatic functions through kinetic and Fd-binding analyses. The K(m) value obtained by assay for a diaphorase activity indicated that R-FNR had a 10-fold higher affinity for NADPH than L-FNR I. When we assayed for NADPH-cytochrome c reductase activity using maize photosynthetic Fd (Fd I) and non-photosynthetic Fd (Fd III), the R-FNR showed a marked difference in affinity between these two Fd isoproteins; the K(m) for Fd III was 3.0 microM and that for Fd I was 29 microM. Consistent with this, the dissociation constant for the R-FNR:Fd III complex was 10-fold smaller than that of the R-FNR:Fd I complex. This differential binding capacity was confirmed by an affinity chromatography of R-FNR on Fd-sepharose with stronger binding to Fd III. L-FNR I showed no such differential interaction with Fd I and Fd III. These data demonstrated that R-FNR has the ability to discriminate between these two types of Fds. We propose that the stronger interaction of R-FNR with Fd III is crucial for an efficient electron flux of NADPH-FNR-Fd cascade, thus supporting Fd-dependent metabolism in non-photosynthetic organs.
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PMID:Differential interaction of maize root ferredoxin:NADP(+) oxidoreductase with photosynthetic and non-photosynthetic ferredoxin isoproteins. 1088 53

The mammalian purified dispersed NADH-ubiquinone oxidoreductase (Complex I) and the enzyme in inside-out submitochondrial particles are known to be the slowly equilibrating mixture of the active and de-activated forms (Vinogradov, A. D. (1998) Biochim. Biophys. Acta 1364, 169-185). We report here the phenomenon of slow active/de-active transition in intact mitochondria where the enzyme is located within its natural environment being exposed to numerous mitochondrial matrix proteins. A simple procedure for permeabilization of intact mitochondria by channel-forming antibiotic alamethicin was worked out for the "in situ" assay of Complex I activity. Alamethicin-treated mitochondria catalyzed the rotenone-sensitive NADH-quinone reductase reaction with exogenousely added NADH and quinone-acceptor at the rates expected if the enzyme active sites would be freely accessible for the substrates. The matrix proteins were retained in alamethicin-treated mitochondria as judged by their high rotenone-sensitive malate-cytochrome c reductase activity in the presence of added NAD(+). The sensitivity of Complex I to N-ethylmaleimide and to the presence of Mg(2+) was used as the diagnostic tools to detect the presence of the de-activated enzyme. The NADH-quinone reductase activity of alamethicin-treated mitochondria was sensitive to neither N-ethylmaleimide nor Mg(2+). After exposure to elevated temperature (37 degrees C, the conditions known to induce de-activation of Complex I) the enzyme activity became sensitive to the sulfhydryl reagent and/or Mg(2+). The sensitivity to both inhibitors disappeared after brief exposure of the thermally de-activated mitochondria with malate/glutamate, NAD(+), and cytochrome c (the conditions known for the turnover-induced reactivation of the enzyme). We conclude that the slow active/de-active Complex I transition is a characteristic feature of the enzyme in intact mitochondria and discuss its possible physiological significance.
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PMID:Catalytic activity of NADH-ubiquinone oxidoreductase (complex I) in intact mitochondria. evidence for the slow active/inactive transition. 1112 57

The mechanisms of cytotoxicity of polynitroaromatic explosives, an important group of environmental pollutants, remain insufficiently studied so far. We have found that the rate constants of single-electron enzymatic reduction, and the enthalpies of single-electron reduction of nitroaromatic compounds (DeltaHf(ArNO(2)(-*)), obtained by quantum mechanical calculation, may serve as useful tools for the analysis of cytotoxicity of nitroaromatic explosives with respect to the possible involvement of oxidative stress. The single-electron reduction rate constants of a number of explosives including 2,4,6-trinitrotoluene (TNT) and 2,4,6-trinitrophenyl-N-methylnitramine (tetryl), and model nitroaromatic compounds by ferredoxin:NADP(+) reductase (FNR, EC 1.18.1.2) and NADPH:cytochrome P-450 reductase (P-450R, EC 1.6.2.4) increased with a decrease in DeltaHf(ArNO(2)(-*)). This indicates that the reduction rates are determined by the electron transfer energetics, but not by the particular structure of the explosives. The cytotoxicity of explosives to bovine leukemia virus-transformed lamb kidney fibroblasts (line FLK) increased with a corresponding increase in their reduction rate constant by P-450R and FNR, or with a decrease in their DeltaHf(ArNO(2)(-*)). This points to an importance of oxidative stress in the toxicity of explosives in this cell line, which was further evidenced by the protective effects of desferrioxamine and the antioxidant N,N'-diphenyl-p-phenylene diamine, and an increase in lipid peroxidation. DT-diaphorase (EC 1.6.99.2) exerted a minor and equivocal role in the cytotoxicity of explosives to FLK cells.
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PMID:Quantitative structure-activity relationships in enzymatic single-electron reduction of nitroaromatic explosives: implications for their cytotoxicity. 1151 95

Because of the evidence for the involvement of xenobiotic bioactivation in pulmonary toxicity and carcinogenesis, it is important to improve our understanding of the xenobiotic-metabolizing enzymes in isolated and cultured specific pulmonary cell populations. Some phase I and phase II xenobiotic-metabolizing enzyme activities, reduced glutathione (GSH), and gamma-glutamyl transferase (gamma-GT) were studied in rat type II pneumocytes and alveolar macrophages cultured for up to 48 h and 3 h, respectively. In type II pneumocytes, 7-ethoxyresorufin activity was not detected. 7-Benzyloxyresorufin (BROD) and 7-pentoxyresorufin (PROD) O-dealkylation decreased at 24 h by 84 and 82%, respectively, and continued to decline over the next 24 h with no measurable PROD at 48 h. The activity of NADPH- and NADH-cytochrome c reductase at 48 h decreased by 31 and 67%, respectively. GST activity decreased by 25 and 42% at 24 and 48 h, respectively. A transient increase in DT-diaphorase activity was observed at 24 h (by 55%). GSH content and gamma-GT activity increased significantly with time in culture. In freshly isolated alveolar macrophages, BROD activity was the only cytochrome P450-dependent alkoxyresorufin-O-dealkylase activity measured. BROD activity decreased by 38% in 3-h-attached macrophages. There were no changes in NADPH- and NADH-cytochrome c reductase, GST, and DT-diaphorase. An increase of GSH (by 24%) was observed in attached macrophages. In conclusion, type II pneumocytes and to a lesser extent alveolar macrophages in primary cultures undergo changes in biotransformation-related enzyme activities and intracellular GSH level that may affect xenobiotic toxicity at different times in culture.
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PMID:Xenobiotic-metabolizing enzyme activities in primary cultures of rat type II pneumocytes and alveolar macrophages. 1156 Aug 80

The gene fprA of Mycobacterium tuberculosis, encoding a putative protein with 40% identity to mammalian adrenodoxin reductase, was expressed in Escherichia coli and the protein purified to homogeneity. The 50-kDa protein monomer contained one tightly bound FAD, whose fluorescence was fully quenched. FprA showed a low ferric reductase activity, whereas it was very active as a NAD(P)H diaphorase with dyes. Kinetic parameters were determined and the specificity constant (kcat/Km) for NADPH was two orders of magnitude larger than that of NADH. Enzyme full reduction, under anaerobiosis, could be achieved with a stoichiometric amount of either dithionite or NADH, but not with even large excess of NADPH. In enzyme titration with substoichiometric amounts of NADPH, only charge transfer species (FAD-NADPH and FADH2-NADP+) were formed. At NADPH/FAD ratios higher than one, the neutral FAD semiquinone accumulated, implying that the semiquinone was stabilized by NADPH binding. Stabilization of the one-electron reduced form of the enzyme may be instrumental for the physiological role of this mycobacterial flavoprotein. By several approaches, FprA was shown to be able to interact productively with [2Fe-2S] iron-sulfur proteins, either adrenodoxin or plant ferredoxin. More interestingly, kinetic parameters of the cytochrome c reductase reaction catalyzed by FprA in the presence of a 7Fe ferredoxin purified from M. smegmatis were determined. A Km value of 30 nm and a specificity constant of 110 microM(-1) x s(-1) (10 times greater than that for the 2Fe ferredoxin) were determined for this ferredoxin. The systematic name for FprA is therefore NADPH-ferredoxin oxidoreductase.
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PMID:Mycobacterium tuberculosis FprA, a novel bacterial NADPH-ferredoxin reductase. 1207 65


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