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)

An enzyme (NADPH-dependent diaphorase) present in rat brain microsomes has been solubilised and shown to utilise both nitrobluetetrazolium and cytochrome c as electron acceptors, when reduced by NADPH. The kinetics of the enzyme have been determined using cytochrome c (Km = 1.3 microM), NADPH (Km = 1.4 microM) and the Vmax (4.7 nmol/min/mg solubilised microsome protein). The subunit Mr is approximately 73,000 D and that of the native enzyme is 170,000-180,000 D, indicating that the enzyme is probably a dimer. Evidence is also provided to show that the enzyme is a flavoprotein, and that it has equimolar amounts of FAD and FMN with respect to the subunit concentration. It seems a possibility that the rat brain diaphorase enzyme may be cytochrome P450 reductase, EC 1.6.2.4.
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PMID:Rat brain NADPH-dependent diaphorase. A possible relationship to cytochrome P450 reductase. 313 10

The activities of UDP-glucuronyl transferase, DT-diaphorase, epoxide hydrolase, aryl hydrocarbon hydroxylase, gamma-glutamyl transferase and NADPH-cytochrome c reductase were measured in the nuclear and microsomal fractions from normal rat liver and rat liver nodules. Nodules were produced by intermittent feeding of Wistar rats with a standard diet supplemented with 0.05% (w/w) 2-acetylaminofluorene. The nuclear and microsomal fractions were isolated by differential centrifugation. The activities of UDP-glucuronyl transferase, DT-diaphorase, epoxide hydrolase and gamma-glutamyl transferase were significantly increased in the nuclear and microsomal fractions obtained from nodules as compared with normal liver. Aryl hydrocarbon hydroxylase activity was decreased in the microsomal fraction from the pathological tissue but not in the nuclear fraction. NADPH-cytochrome c reductase activity was similar in nodular and normal liver tissue. The nuclear/microsomal ratio for phase I reactions in xenobiotic metabolism was increased over normal more than two fold. Thus the nuclear and microsomal systems for drug metabolism are both changed in liver nodules. The relative enhancement of nuclear activating reactions is remarkable in the light of the increased risk for malignant transformation exhibited by nodular cells.
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PMID:Profile of drug metabolizing enzymes in the nuclear and microsomal fractions from rat liver nodules and normal liver. 324 42

The interaction of N-(4-ethoxyphenyl)p-benzoquinone imine (NEPBQI), a metabolite formed during peroxidase catalyzed metabolism of p-phenetidine, with GSH and its effects in isolated rat hepatocytes were investigated. When reacted with GSH NEPBQI formed both a mono- and a diglutathione conjugate as well as GSSG. Formation of glutathione conjugates and GSSG also occurred when NEPBQI was added to isolated hepatocytes. The formation of GSSG was, however, only detectable if the hepatocytes had been pretreated with the GSSG reductase inhibitor BCNU (1,3-bis-(2-chloroethyl-1-nitrosourea). Similarly, NEPBQI caused a rapid decrease in cellular free protein thiols when added to hepatocytes, however this was expressed at higher concentrations than for effects on GSH. The protein thiol decrease was correlated with protein binding of NEPBQI. NEPBQI was also shown to be toxic to isolated hepatocytes. At a concentration of 400 microM extensive bleb formation was followed by loss of cell membrane integrity and cell death. To assess further the subcellular metabolism of NEPBQI microsomes and cytosol was used. NEPBQI was found to be preferentially reduced by cytochrome P-450 reductase in the microsomes whereas DT-diaphorase catalyzed its reduction in cytosol. NEPBQI did not undergo significant redox cycling since no formation of O2 was observed. Thus, the cytotoxic effect of NEPBQI appears to be due to protein arylation rather than redox cycling.
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PMID:Cellular effects of N(4-ethoxyphenyl)p-benzoquinone imine, a p-phenetidine metabolite formed during peroxidase reactions. 379 94

Since the cure of solid tumors is limited by the presence of cells with low oxygen contents, we have approached the development of treatment regimens and of new drugs for these tumors by investigating agents which are preferentially bioactivated under hypoxia. Major emphasis has been directed at studying the mode of action of the mitomycin antibiotics, as bioreductive alkylating agents. Using primarily the EMT6 mouse mammary carcinoma as a solid tumor model, we have found that mitomycin C and porfiromycin are preferentially toxic to cells with low oxygen contents. The mitomycin analog BMY-25282 is more toxic to hypoxic cells than are mitomycin C and porfiromycin; however, unlike these antibiotics, BMY-25282 is preferentially toxic to well-oxygenated cells. With these three mitomycins, we have observed a correlation between cytotoxicity to hypoxic cells, the rate of generation of reactive products, and the redox potentials of the drugs. Investigations of the enzymes in EMT6 cells that could possibly activate mitomycin C have revealed that cytochrome P-450 and xanthine oxidase are not present in measurable quantities and therefore are not responsible for activation of mitomycin C. Activities representative of NADPH-cytochrome c reductase and DT-diaphorase are present in these neoplastic cells. Comparison of these enzymatic activities in EMT6, CHO, and V79 cells with the rate of generation of reactive products under hypoxia shows a direct correlation between these two parameters, but there is no quantitative correlation between these two parameters and the amount of cytotoxicity. Use of purified NADPH-cytochrome c reductase and inhibitors of this enzyme demonstrated that NADPH-cytochrome c reductase can activate mitomycin C, but that it is probably not the only enzyme participating in this bioactivation in EMT6 cells. The DT-diaphorase inhibitor dicoumarol was employed to show that this enzyme is not involved in the activation of mitomycin C to a cytotoxic agent. Instead, DT-diaphorase appears to metabolize mitomycin C to a nontoxic product. This property has been exploited to develop a new treatment regimen for solid tumors. Using X-rays to eliminate well oxygenated cells of a solid tumor implant of the EMT6 carcinoma, we have found that the combination of dicoumarol plus mitomycin C is more toxic to hypoxic tumor cells in vivo than mitomycin C alone. Furthermore, knowledge of the biochemical mechanism of mitomycin C activation permits a prediction of which tumors can best be treated with this combination of drugs by measuring enzymatic activities in biopsy specimens.
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PMID:Chemotherapeutic attack of hypoxic tumor cells by the bioreductive alkylating agent mitomycin C. 393 22

Reduced nicotinamide adenine dinucleotide (NADH):ferricyanide reductase and DT-diaphorase specific activity in total homogenates of rat liver are markedly decreased as a very early biochemical event of hepatocarcinogenesis induced by the carcinogen 2-acetylaminofluorene (AAF). A 50 to 75% decrease in NADH:ferricyanide reductase was observed after 1 day of AAF (0.025% in the diet) feeding and persisted throughout a 7-week continuum of AAF administration. Carcinogen added directly to cell extracts had no effect. Similar results were obtained with single injections of either AAF or diethylnitrosamine. Xanthine dehydrogenase was also reduced in liver following AAF administration to nearly the same extent as NADH:ferricyanide reductase and DT-diaphorase. Total NADH-cytochrome c reductase and mitochondrial activity as estimated from succinic dehydrogenase were not affected by carcinogen administration relative to basal dietary controls. The reduced nicotinamide adenine dinucleotide phosphate:cytochrome c reductase that functions in drug detoxification was elevated. With livers of animals fed 4-acetamidophenol, a hepatotoxin chemically related to AAF, small decreases were noted in NADH:ferricyanide reductase, but not in xanthine dehydrogenase nor in DT-diaphorase. Initial lowering of these activities in the livers of the carcinogen-treated animals is preceded by or concomitant with a reduction in the levels of extramitochondrial pyridine nucleotides known from other studies to result from DNA damage.
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PMID:Decreased NADH-oxidoreductase activities as an early response in rat liver to the carcinogen 2-acetylaminofluorene. 396 29

Monodehydroascorbate reductase (EC 1.6.5.4) was purified from cucumber fruit to a homogeneous state as judged by polyacrylamide gel electrophoresis. The cucumber monodehydroascorbate reductase was a monomer with a molecular weight of 47,000. It contained 1 mol of FAD/mol of enzyme which was reduced by NAD(P)H and reoxidized by monodehydroascorbate. The enzyme had an exposed thiol group whose blockage with thiol reagents inhibited the electron transfer from NAD(P)H to the enzyme FAD. Both NADH and NADPH served as electron donors with Km values of 4.6 and 23 microM, respectively, and Vmax of 200 mol of NADH and 150 mol of NADPH oxidized mol of enzyme-1 s-1. The Km for monodehydroascorbate was 1.4 microM. The amino acid composition of the enzyme is presented. In addition to monodehydroascorbate, the enzyme catalyzed the reduction of ferricyanide and 2,6-dichloroindophenol but showed little reactivity with calf liver cytochrome b5 and horse heart cytochrome c. The kinetic data suggested a ping-pong mechanism for the monodehydroascorbate reductase-catalyzed reaction. Cucumber monodehydroascorbate reductase occurs in soluble form and can be distinguished from NADPH dehydrogenase, NADH dehydrogenase, DT diaphorase, microsome-bound NADH-cytochrome b5 reductase, and NADPH-cytochrome c reductase by its molecular weight, amino acid composition, and specificity of electron acceptors and donors.
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PMID:Monodehydroascorbate reductase from cucumber is a flavin adenine dinucleotide enzyme. 405 27

1. The values of the protein, RNA and phospholipid concentrations within the total microsomal fractions obtained from different stages of embryonic chick liver are compared. 2. Only the phospholipid content increases significantly with increasing developmental age. 3. The lack of membranes in the early stages of development and the relative constancy of RNA values during development suggests that some of the protein present at the early developmental stages is of a non-membranous non-ribosomal nature. 4. Glucose 6-phosphatase, adenosine triphosphatase, NADH(2)-cytochrome c reductase and diaphorase all increased in activity as development progressed. 5. Comparisons of submicrosomal fractions with respect to their protein, RNA and phospholipid content showed that in all embryonic stages fraction II (rough-membrane fraction) contained more than 60% of the proteins, RNA and phospholipid of the microsomal fraction. 6. Glucose 6-phosphatase was shown to be present predominantly in fraction II, whereas adenosine triphosphatase was present predominantly in fraction Iab (smooth-membrane fraction). 7. The significance of the differences between the smooth- and rough-microsomal fractions is discussed.
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PMID:Changes in the chemical composition and the enzymic activities of hepatic microsomes of the chick embryo during development. 604 89

Rat astrocytes in primary cultures were employed to isolate the plasma membrane. The method for the isolation of plasma membrane was based on the capacity of the cytoskeleton to adhere to the substratum entrapping intracellular organelles during freezing-thawing cycle performed on the cell. By washing the 'surface adherent framework', the untrapped plasma membrane were recovered and density equilibrium centrifugation resulted in the isolated membrane. The isolated plasma membrane was characterized on the basis of a variety of marker enzymes positive to the plasma membrane such as (Na+ + K+)-ATPase or 5'-nucleotidase as well as the lack of conventional markers of other endomembranes. Ultrastructurally the membranes, as isolated here, were mainly vesicular in nature. The isolated plasma membrane was devoid of the dehydrogenase responsible for NADH-cytochrome c reductase activity. However, NADH-ferricyanide reductase activity and the dehydrogenase system catalyzing the transfer of reducing equivalents from NADH or NADPH to dichloroindophenol seems plasma membrane redox system. The identical specific activity employing dichloroindophenol as an electron acceptor with NADH or NADPH as donor indicate a DT-diaphorase (EC 1.6.99.2) like activity in the astrocytes plasma membrane.
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PMID:Plasma membrane isolated from astrocytes in primary cultures. Its acceptor oxidoreductase properties. 609 77

A membrane-associated b-type cytochrome (a proposed component in the neutrophil microbicidal superoxide generating system) has been partially purified from nonactivated beef granulocytes to a specific heme content of 20 nmol of heme/mg of protein, a value about 10-fold higher than those previously reported. The hemoprotein was solubilized at low temperature (4 degrees C) from mixed granule (30,000 X g) cell fractions using Triton X-114 detergent. Warming the extract to 25 degrees C allowed separation into detergent and aqueous phases; cytochrome b558 partitioned exclusively into the detergent phase, allowing separation from other visible-absorbing species (e.g. myeloperoxidase) and indicated an intrinsic membrane localization (Bordier, C. (1981) J. Biol. Chem. 256, 1604-1607). The partitioned cytochrome was chromatographed on hydroxylapatite and a hydrophobic affinity matrix, allowing a 185-fold (heme content) purification from the granule extract. The cytochrome preparation revealed three equal-staining protein bands by sodium dodecyl sulfate-urea polyacrylamide gel electrophoresis; apparent molecular weights were 14,000, 12,000, and 11,000. The question of heterogeneity of the preparation versus subunit structure is not resolved at present. The hemoprotein binds carbon monoxide, consistent with a proposed role as a terminal oxidase, and has an unusually negative oxidation-reduction potential (-225 mV) similar to that observed in granulocyte membranes. The preparation is devoid of NAD(P)H-diaphorase and cytochrome c reductase activities.
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PMID:Cytochrome b558 from (bovine) granulocytes. Partial purification from Triton X-114 extracts and properties of the isolated cytochrome. 643 85

Hypoxic cells of solid tumors are difficult to eradicate by X-irradiation or chemotherapy; as an approach to this problem, our laboratories are investigating the effects of the bioreductive alkylating agent mitomycin C (MC) on hypoxic cells. This antibiotic was preferentially toxic to EMT6 mouse mammary tumor cells and V79 Chinese hamster lung fibroblasts under hypoxic conditions, but it was equitoxic to Chinese hamster ovary cells in the presence and absence of oxygen. All cell lines catalyzed the formation of reactive metabolites under hypoxic conditions and contained NADPH:cytochrome c reductase and DT-diaphorase, two enzymes which may be responsible for the cellular activation of MC. Although a correlation existed between enzymatic activities and the formation of reactive metabolites from MC, there was no correspondence between these parameters and the degree of cytotoxicity expressed by MC under hypoxic conditions. Purified NADPH:cytochrome c reductase reduced MC in the absence of oxygen, with addition of cytochrome P-450 enhancing, but not participating directly in, the reduction reaction. Addition of NADP+ to cell sonicates substantially reduced NADPH:cytochrome c reductase activity, while the formation of reactive metabolites was affected only slightly; converse results were observed using mersalyl. Exposure of cell sonicates to dicumarol inhibited DT-diaphorase activity, while the rate of formation of reactive metabolites of MC was enhanced. The findings suggest that NADPH:cytochrome c reductase and some as yet to be identified enzyme(s) are important for the reductive activation of MC. DT-diaphorase and cytochrome P-450 are not directly involved in the activation of MC, but they appear to modulate the degree of activation to reactive species, which are presumably responsible for the observed cytotoxicity.
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PMID:Role of NADPH:cytochrome c reductase and DT-diaphorase in the biotransformation of mitomycin C1. 643 71


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