Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:1.17.3.2 (xanthine oxidase)
 8,383 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The properties of the interactions of anticancer quinone drugs, aclacinomycin A, adriamycin, carbazilquinone, and mitomycin C with nicotinamide adenine dinucleotide phosphate (NADPH)-cytochrome P-450 reductase and xanthine oxidase under anaerobic and aerobic conditions were studied. Km values of NADPH-cytochrome P-450 reductase for these drugs were in the range of 40-227 microM, and that of deflavo xanthine oxidase in the range of 39-over 200 microM. Under anaerobic conditions, when xanthine was used as an electron donor, deflavo xanthine oxidase catalyzed the reductive glycosidic cleavage reaction of anthracyclines and nicotinamide adenine dinucleotide was ineffective as an electron donor. In the electron spin resonance study, the formation of the semiquinone or free radical state of the quinone drugs in both enzyme systems were evidenced. A weak and symmetric signal was obtained from aclacinomycin A, and a symmetric signal from adriamycin was changed into an asymmetric and strong. The hyperfine structure was obtained from carbazilquinone in the oxidase system. In the studies of ultraviolet-visible spectra of the quinone drugs in the reductase system, the spectra of aclacinomycin A and adriamycin were changed to their 7-deoxylaglycones, and the formation of small amounts of the fully reduced form were observed after long incubations. The spectrum of carbazilquinone was changed to the hydroquinone form and mitomycin C was converted into mitosene analogues. Under aerobic conditions, superoxide radicals and hydrogen peroxide were effectively produced in the presence of anticancer quinone drugs in both enzyme systems. The superoxide-dependent hydroxy radical production, which was measured by ethylene production from methional, was observed in the presence of aclacinomycin A and adriamycin in the deflavo xanthine oxidase system. From these results, the possible reactions in the interactions of anticancer quinone drugs with these enzymes and oxygen are discussed.
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PMID:Interactions of anticancer quinone drugs, aclacinomycin A, adriamycin, carbazilquinone, and mitomycin C, with NADPH-cytochrome P-450 reductase, xanthine oxidase and oxygen. 302

1. The topography of cytochrome P-450 in vesicles from smooth endoplasmic reticulum of rat liver has been examined. Approx. 50% of the cytochrome is directly accessible to the action of trypsin in intact vesicles whereas the remainder is inaccessible and partitioned between luminal-facing or phospholipid-embedded loci. Analysis by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis reveals three major species of the cytochrome. Of these, the variant with a mol.wt. of 52000 is induced by phenobarbitone and this species is susceptible to trypsin. 2. After trypsin treatment of smooth membrane, some NADPH-cytochrome P-450 (cytochrome c) reductase activity remains and this remaining activity is enhanced by treatment with 0.05% deoxycholate, which renders the membranes permeable to macromolecules. In non-trypsin-treated control membranes the reductase activity is increased to a similar extent. These observations suggest an asymmetric distribution of NADPH-cytochrome P-450 (cytochrome c) reductase in the membrane. 3. As compared with dithionite, NADPH reduces only 44% of the cytochrome P-450 present in intact membranes. After tryptic digestion, none of the remaining cytochrome P-450 is reducible by NADPH. 4. In the presence of both a superoxide-generating system (xanthine plus xanthine oxidase) and NADPH, all the cytochrome P-450 in intact membrane (as judged by dithionite reducibility) is reduced. The cytochrome P-450 remaining after trypsin treatment of smooth vesicles cannot be reduced by this method. 5. The superoxide-dependent reduction of cytochrome P-450 is prevented by treatment of the membranes with mersalyl, which inhibits NADPH-cytochrome P-450 (cytochrome c) reductase. Thus the effect of superoxide may involve NADPH-cytochrome P-450 reductase and cytosolically orientated membrane factor(s).
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PMID:Asymmetric distribution of cytochrome P-450 and NADPH--cytochrome P-450 (cytochrome c) reductase in vesicles from smooth endoplasmic reticulum of rat liver. 625 76

Under anaerobic conditions and with proper electron donors, NADPH-cytochrome P-450 reductase (EC 1.6.2.4) and xanthine oxidase (EC 1.2.3.2) similarly reductively metabolized mitomycin C. Reversed phase high performance liquid chromatography was used to separate, detect, and isolate several metabolites. Three metabolites were identified by mass spectrometry and thin layer chromatography as 1,2-cis- and trans-2,7-diamino-1-hydroxymitosene and 2,7-diaminomitosene. Three metabolites were phosphate-dependent, and two of them were identified to be 1,2-cis- and trans-2,7-diaminomitosene 1-phosphate. The amounts of the five identified metabolites generated during the reduction of mitomycin C varied with pH and nucleophile concentration. At pH 6.5, 2,7-diaminomitosene was essentially the only metabolite formed, whereas from pH 6.8 to 8.0, trans- and cis-2,7-diamino-1-hydroxymitosene increased in quantity as 2,7-diaminomitosene decreased. The disappearance of mitomycin C and the production of metabolites were enzyme and mitomycin C concentration-dependent. Substrate saturation was not reached for either enzyme up to 5 mM mitomycin C. Electron paramagnetic resonance studies demonstrated the formation of mitomycin C radical anion as an intermediate during enzymatic activation. Our results indicate that either enzyme catalyzed the initial activation of mitomycin C to a radical anion intermediate. Subsequent spontaneous reactions, including the elimination of methanol and the opening of the aziridine ring, generate one active center at C-1 which facilitates nucleophilic attack. Simultaneous generation of two reactive centers was not observed. All five primary metabolites were metabolized further by either flavoenzyme. The secondary metabolites exhibited similar changes in their absorbance spectra and were unlike the primary metabolites, suggesting that a second alkylating center other than C-1 was generated during secondary activation. We propose that secondary activation of monofunctionally bound mitomycin C is probably a main route for the bifunctional binding of mitomycin C to macromolecules and that the cytotoxic actions of mitomycin C result from multiple metabolic activations and reactions.
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PMID:Reductive activation of mitomycin C and mitomycin C metabolites catalyzed by NADPH-cytochrome P-450 reductase and xanthine oxidase. 631 93

Benzphetamine demethylase and aniline hydroxylase activities were determined with various hemoproteins including indoleamine 2,3-dioxygenase in a cytochrome P-450-like reconstituted system containing NADPH, NADPH-cytochrome P-450 reductase, and O2. The highest specific activities, almost comparable to those of liver microsomal cytochrome P-450, were detected with indoleamine 2,3-dioxygenase from the rabbit intestine. The indoleamine 2,3-dioxygenase-catalyzed benzphetamine demethylation reaction was inhibited by catalase but not by superoxide dismutase. Exogenous H2O2 or organic hydroperoxides was able to replace the reducing system and O2. The stoichiometry of H2O2 added to the product formed was essentially unity. These results indicate that the dioxygenase catalyzes the demethylation reaction by the so-called "peroxygenation" mechanism using H2O2 generated in the reconstituted system. On the other hand, the dioxygenase-catalyzed aniline hydroxylation reaction was not only completely inhibited by catalase but also suppressed by superoxide dismutase by about 60%. Although the O2- and H2O2-generating system (e.g. hypoxanthine-xanthine oxidase) was also active as the reducing system, neither exogenous H2O2 nor the generation of O2- in the presence of catalase supported the hydroxylation reaction, indicating that both H2O2 and O2- were essential for the hydroxylation reaction. However, typical scavengers for hydroxyl radical and singlet oxygen were not inhibitory. These results suggest that a unique, as yet unidentified active oxygen species generated by H2O2 and O2- participates in the dioxygenase-mediated aniline hydroxylation reaction.
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PMID:Monooxygenase activities of dioxygenases. Benzphetamine demethylation and aniline hydroxylation reactions catalyzed by indoleamine 2,3-dioxygenase. 640 89

The mechanism of cytochrome P-450-dependent oxidation of ethanol has been investigated using reconstituted phospholipid vesicles containing purified preparations of rabbit liver microsomal NADPH-cytochrome P-450 reductase and cytochrome P-450 LM2. Incorporation of cytochrome b5 into the vesicles resulted in a 5-fold enhancement of cytochrome P-450-catalyzed O-dealkylation of 7-ethoxycoumarin, whereas the cytochrome P-450-dependent ethanol oxidation was slightly inhibited. Superoxide dismutase, added in increasing amounts to the vesicles, inhibited the formation of superoxide anions and, in a concomitant manner, also the production of acetaldehyde from ethanol in the system. Also horseradish peroxidase inhibited ethanol oxidation catalyzed by the vesicles; acetaldehyde formation and H2O2 formation decreased in a concomitant manner as the amount of the peroxidase was increased. Externally added hydrogen peroxide markedly stimulated cytochrome P-450-dependent ethanol oxidation, but not until the concentration of H2O2 reached 0.3 mM, whereas the hydroxyl radical scavenger mannitol completely inhibited the cytochrome P-450-dependent acetaldehyde production. Oxidation of ethanol was also accomplished using vesicles containing cytochrome b5 instead of cytochrome P-450 and in other systems regenerating superoxide anions, e.g. the xanthine-xanthine oxidase system and dihydroxyfumarate. The results are consistent with an iron-catalyzed Haber-Weiss mechanism for regeneration of hydroxyl radicals which subsequently react with ethanol, thereby giving the corresponding aldehyde.
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PMID:The mechanism of cytochrome P-450-dependent oxidation of ethanol in reconstituted membrane vesicles. 678 51

Purified liver microsomal NADPH-cytochrome P-450 reductase is able to catalyze the activation of [14C]ronidazole to metabolite(s) which bind covalently to protein. Like the reaction catalyzed by microsomes, protein alkylation catalyzed by the reductase is (1) sensitive to oxygen, (2) requires reducing equivalents, (3) is inhibited by sulfhydryl-containing compounds and (4) is stimulated several fold by either flavin mononucleotide (FMN) or methytlviologen. A cytochrome P-450 dependent pathway of ronidazole activation can be demonstrated as judged by the inhibition of the reaction by carbon monoxide, metyrapone and 2,4-dichloro-6-phenylphenoxyethylamine but the involvement of specific microsomal cytochrome P-450 isozymes has not been definitively established. Milk xanthine oxidase is also capable of catalyzing ronidazole activation. Polyacrylamide sodium dodecyl sulfate (SDS)-gel electrophoresis reveals that the reactive intermediate(s) of ronidazole does not alkylate proteins selectively.
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PMID:Drug residue formation from ronidazole, a 5-nitroimidazole. II. Involvement of microsomal NADPH-cytochrome P-450 reductase in protein alkylation in vitro. 680 46

1. The subcellular distribution of nitrobenzene reduction activity in rat liver cells indicated the existence of two different enzyme systems, one localized in microsomes and the other localized in cytosol. The activity in the cytosol was mainly attributable to xanthine oxidase, judging from its substrate specificity and the inhibition by allopurinol. 2. The participation of the microsomal electron transport system in nitrobenzene reduction was examined by using antibodies against four components of the system, NADPH-cytochrome c reductase (fpT), NADH-cytochrome b5 reductase (fpD), cytochrome b5, and cytochrome P-450. Both NADH- and NADPH-dependent nitrobenzene reduction activities were strongly inhibited by anti-fpT IG and also by anti-P450 IG, but not inhibited by anti-fpD IG or anti-b5 IG. The reduction of nitrosobenzene and phenylhydroxylamine, which are supposed to be the intermediates of nitrobenzene reduction, was also examined, and it was found that NADH- and NADPH-dependent reduction of both compounds were strongly inhibited by anti-fpT IG and anti-P450 IG, but not by anti-fpD IG or anti-b5 IG. 3. Reconstruction experiments using purified NADPH-cytochrome P-450 reductase and cytochrome P-450 were also carried out and it was confirmed that the reduction of nitrobenzene, nitrosobenzene, and phenylhydroxylamine to aniline could be effected by these two components. 4. Nitrobenzene reduction by microsomes exhibited a short initial time lag and was activated by the addition of purified NADPH-cytochrome c reductase, whereas nitrosobenzene and phenylhydroxylamine reductions did not show any initial time lag and were not activated by the reductase. These observations suggest that the reduction of nitrobenzene to an intermediate, possibly nitrosobenzene or phenylhydroxylamine, limits the rate of aniline formation, and such an initial step of nitrobenzene reduction can be catalyzed by NADPH-cytochrome c reductase alone. Cytochrome P-450 is essential at least in the final step of nitrobenzene reduction to aniline. This conclusion was further confirmed by determination of these intermediates in nitrobenzene reduction.
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PMID:Participation of cytochrome P-450 in the reduction of nitro compounds by rat liver microsomes. 739 Sep 98

The characterization of the enzymatic step(s) involved in the reduction of 3'-azido-3'-deoxythymidine (zidovudine)(ZDV) to 3'-amino-3'-deoxythymidine (AMT) was pursued. AMT formation by human liver microsomes was NADPH dependent, enhanced under anaerobic conditions, and increased by flavin adenine dinucleotide (FAD) and FMN. Carbon monoxide inhibited AMT formation by up to 80%. The effect of theophylline (CYP1A substrate), tolbutamide (CYP2C substrate), chlorzoxazone, thiobenzamide, p-nitrophenol, mercaptoethanol, isoniazid (CYP2E substrates), cortisol (CYP3A substrate), ketoconazole, itraconazole, fluconazole, cimetidine, micronazole (CYP inhibitors), methimazole (flavin-containing mono-oxygenase inhibitor), chloramphenicol (undergoes nitroreduction), allopurinol (xanthine oxidase inhibitor) and dicoumarol (DT-diaphorase inhibitor) on AMT formation were studied to see if the reduction reaction was mediated by a particular isozyme. The greatest inhibition was observed with ketoconazole (concentration producing 50% inhibition = 78.0 microM). At this concentration ketoconazole acted as a non-selective inhibitor of several CYP isozymes. Overall, these data suggested that ZDV reduction was probably mediated by both cytochrome P450 isozymes and NADPH-cytochrome P450 reductase. Formation of AMT, as measured by intrinsic clearance (Clint), was significantly increased in microsomes from rats pre-treated with phenobarbitone, dexamethasone and clofibrate (inducers of CYP2B, CYP3A and CYP4A, respectively). Pre-treatment of rats with beta-naphthoflavone and ethanol (CYP1A and CYP2E1 inducers, respectively) had no effect on AMT formation.
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PMID:The metabolism of zidovudine by human liver microsomes in vitro: formation of 3'-amino-3'-deoxythymidine. 805 24

The oxidation rate of NADPH is markedly stimulated during the mechanism-based inactivation of cytochrome P450 2B1 by N-methylcarbazole (NMC) in a reconstituted system consisting of NADPH-cytochrome P450 reductase, cytochrome P450 and phospholipid. The stimulation of NADPH oxidation in this system is due to 1-hydroxy-N-methylcarbazole (1-OH-NMC), one of the major metabolites of NMC. The 1-OH-NMC is further metabolized in an NADPH-dependent manner by the reconstituted system or by purified NADPH-cytochrome P450 reductase to give a more polar metabolite which has been isolated by HPLC. The conversion of 1-OH-NMC to this product was inhibited by superoxide dismutase (SOD), and incubation of the 1-OH-NMC with hypoxanthine-xanthine oxidase resulted in the formation of the same product, suggesting that the superoxide anion was involved in the metabolism of 1-OH-NMC by the reductase. Redox cycling activity during the metabolism of 1-OH-NMC by reductase has been demonstrated. The oxidation of NADPH by the reductase in the presence of 35 microM 1-OH-NMC was enhanced approximately 23-fold [240 nmol of NADPH oxidized/(min.nmol of reductase)] relative to control levels in the presence of 500 microM NMC [10.5 nmol/(min.nmol of reductase)]. 1-OH-NMC (35 microM) caused a 40-fold increase in the rate of formation of superoxide during its metabolism by reductase. The rapid rates of NADPH oxidation and superoxide formation were inhibited by the addition of reduced glutathione (GSH) to the reaction mixture. Neither SOD nor GSH inhibited the reductase activity directly.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:The mechanism of stimulation of NADPH oxidation during the mechanism-based inactivation of cytochrome P450 2B1 by N-methylcarbazole: redox cycling and DNA scission. 819 13

DNA adduct formation by enzyme-activated antibiotics, mitomycin C (MMC) or porfiromycin (PFM), at pH 7.6 or pH 6.0 under anaerobic conditions was analyzed by a 32P-postlabeling method. Antibiotic activation by rat liver NADPH-cytochrome P-450 reductase (EC 1.6.2.4) and bovine milk xanthine oxidase (EC 1.2.3.2) produced similar results. Five 32P-labeled MMC adducts were separated by thin layer chromatography and high performance liquid chromatography from DNA alkylated at either pH. Four of the radioactive spots separated by thin layer chromatography were identified as two monofunctional monoadducts [1" alpha and 1" beta forms of N2-(2" beta,7"-diaminomitosen-1"-yl)-2'-deoxyguanylic acid], one bifunctional monoadduct [N2-(10"-decarbamoyl-2",7"-diaminomitosen-1" alpha-yl)-2'-deoxyguanylic acid], and one cross-linked adduct [N2-(2" beta,7"-diamino-10"-deoxyguanyl-N2-yl-mitosen- 1" alpha-yl)-2'-deoxyguanylic acid]. One minor radioactive spot was not identified. By comparing DNA alkylated at the two pH values, based on equal amounts of 32P radioactivity, similar amounts of cross-links were detected. However, the DNA showed different ratios of the alpha and beta isomers of the monofunctional monoadduct. Furthermore, the DNA alkylated at pH 6.0 showed more bifunctional monoadducts than did the DNA alkylated at pH 7.6. Analysis of alkylated DNA by enzyme-activated PFM showed a similar spectrum of DNA adduct formation. The effect of pH on the distribution of the five PFM-DNA adducts was similar to that observed for the five MMC-DNA adducts. The distribution of adducts in DNA alkylated at the same pH was similar irrespective of which enzyme activated MMC or PFM. The pH of the reaction during DNA and MMC interaction was the determining factor for the quantitative distribution of the adducts. This pH effect may be important for the cytotoxicity of MMC and PFM in tumor cells that have high levels of reductive enzymes with low optimal pH values.
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PMID:Effect of pH on DNA alkylation by enzyme-activated mitomycin C and porfiromycin. 839 Nov 16


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