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:1.6.99.3 (diaphorase)
5,903 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Plasma membranes, contaminated very little by endoplasmic reticulum elements, were isolated from rat liver. Activities of NADH-oxidoreductase and b-type cytochromes were higher than expected from this contamination. Between 50 and 80% of these activities were endogenous to plasma membrane. A similar result was obtained after refractionation of digitonin-treated plasma membranes on a sucrose density gradient. This is further evidence for the localization of NADH-ferricyanide reductase, NADH-cytochrome c reductase, and cytochromes b5, P450 and P420 in liver cell membranes.
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PMID:Density gradient fractionation of digitonin-treated rat liver plasma membranes and subcellular localization of NADH-oxidoreductase and B-type cytochromes. 710 80

The distribution of nitric oxide synthase (NOS) in the mouse olfactory bulb and olfactory epithelium, including the vomeronasal organ, was studied using an anti-NOS antibody, NADPH diaphorase histochemistry and in situ hybridization with NOS specific antisense oligonucleotide probes. Interneurons containing NOS protein and mRNA, and exhibiting NADPH diaphorase activity were detected in the plexiform layer of the main olfactory bulb and the granule cell layer of main and accessory olfactory bulbs. Periglomerular cells and granule cells in the main olfactory bulb were also NOS positive with diaphorase and immunostaining for NOS. In contrast, no evidence for NOS expression was found either in the main olfactory epithelium or in the vomeronasal organ, in spite of the strong diaphorase staining of the surface of the main olfactory epithelium. Polymerase chain reaction amplification experiments for detection of NOS gene expression further indicated that NOS is expressed in the olfactory bulb but not in either the main olfactory epithelium or vomeronasal organ. Use of an antibody raised against another enzyme, NADPH-P450 oxidoreductase, showed that this protein was strongly expressed in the olfactory epithelium. Activity of this enzyme may account for the diaphorase histochemical staining of the epithelia. An involvement of neuronal nitric oxide synthase in signalling in olfactory receptor neurons is therefore doubtful, although NOS is clearly expressed in neurons in both main and accessory olfactory bulbs.
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PMID:Localization of nitric oxide synthase in the mouse olfactory and vomeronasal system: a histochemical, immunological and in situ hybridization study. 751 Feb 6

While acute lindane treatment and chronic ethanol feeding to rats have been associated with hepatic oxidative stress, the possible roles of these stresses in the pathogenesis of hepatic lesions reported in acute lindane intoxication and in those observed in some models of chronic alcoholism have not been established. Our previous studies in rats chronically fed ethanol regimens and then treated with a single intraperitoneal (i.p.) dose of lindane (20 mg/kg) showed that while lindane per se was invariably associated with hepatic oxidative stress, chronic ethanol feeding only produced this stress when the dietary level of vitamin E was relatively low. Chronic ethanol pretreatment did not significantly affect the lindane-associated oxidative stress, and neither chronic ethanol feeding nor acute lindane, single or in combination, produced any histologic and biochemical evidence of liver damage. In the present experiment, the acute dose of lindane was increased to 40 mg/kg, and we have studied a larger number of prooxidant and antioxidant hepatic factors. Male Wistar rats (115.5 +/- 5.4 g) were fed ad lib for 11 weeks a calorically well-balanced and nutritionally adequate basal diet, or the same basal diet plus a 32% ethanol/25% sucrose solution, also ad lib, and were then injected i.p. with a single dose of lindane or with equivalent amounts of corn oil. The results indicated that acute lindane treatment to naive rats increased practically all the prooxidant hepatic factors examined (cytochromes P450 and b5, NADPH cytochrome c reductase, NADPH oxidase), as well as the generation of microsomal superoxide radical and thiobarbituric acid reactive substances of liver homogenates, but did not modify any of the antioxidant hepatic factors studied. Conversely, the chronic administration of ethanol alone did not significantly affect the prooxidant hepatic factors but reduced some of the antioxidants (i.e., the activities of GSH-Px and the contents of alpha-tocopherol and ubiquinols 9 and 10). Although chronic ethanol pretreatment further increased the superoxide generation induced by lindane per se, it did not increase but generally reduced the effects of lindane per se on the other prooxidant factors studied. Furthermore, although acute lindane administration to ethanol-pretreated rats was associated with decreases in GSH and catalase (not affected by ethanol or lindane treatment alone), it did not substantially modify the reducing effects of ethanol feeding per se on GSH-Px, alpha-tocopherol, and ubiquinols. Once again, neither chronic ethanol feeding nor lindane treatment, single or in combination, was associated with any evidence of liver damage.
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PMID:Prooxidant and antioxidant hepatic factors in rats chronically fed an ethanol regimen and treated with an acute dose of lindane. 754 17

A metallothionein-I-transgenic mouse strain (MT-TG) was characterized to determine whether they would be suitable to study the functions of this protein. MT-TG mice were visually indistinguishable from nontransgenic littermate controls, but had 10- to 20-fold higher basal levels of MT protein in pancreas, liver, and stomach, as well as 2- to 6-fold higher MT protein levels in other organs (kidney, intestine, uterus, testes, spleen, heart, and lung) than control mice, as determined by the Cd/hemoglobin assay. The MT-TG mice had 50% more Zn in liver and 300% more Zn in pancreas than control mice. Interestingly, female MT-TG mice have 4- to 5-fold higher MT levels in liver than those of males. To determine whether MT can be further increased by well-known MT inducers, control and MT-TG mice were given Zn (200 mumol/kg), Cd (20 mumol/kg), or diethyl maleate (DEM, 5 mmol/kg), and tissue MT concentrations were measured 24 hr later. MT-TG mice responded to MT inducers in a manner similar to control mice. The hepatic antioxidant components (glutathione (GSH), GSH-peroxidase, GSH-reductase, GSH S-transferase, superoxide dismutase, DT-diaphorase, and catalase) of MT-TG mice were not different from those of controls. The cytochrome P450 enzymes (total P450, b5, NADPH cytochrome c reductase) were normal in these MT-TG mice. The activities of CYP1A, CYP2B, and CYP2E enzymes in MT-TG mice were also similar to those of controls, as determined by ethoxy- and pentoxyresorufin O-dealkylation and chlorzoxazone 6-hydroxylation. Thus, MT-TG mice appear to be a good model for studying functions of MT.
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PMID:Characterization of metallothionein-I-transgenic mice. 764 27

Human cytochrome CYP3A4 is the most abundant of all the P450s in human liver and is involved in the metabolism of many environmental toxicants and drugs. Kinetic studies with CYP3A4 have been hampered due to low activity of this enzyme obtained from recombinant gene expression systems or difficulty in reconstituting activity with the native enzyme purified from human liver. To overcome these obstacles, we have expressed high levels of catalytically active CYP3A4 and human NADPH-cytochrome P450 reductase (CYPOR) together in two insect cell lines, Spodoptera frugiperda (Sf9) and Trichoplusia ni (T.ni), via a single recombinant baculovirus carrying both cDNAs (CYP3A4-OR). Microsomes containing recombinant CYP3A4-OR from these cell lines were up to 50-times more active in testosterone 6 beta-hydroxylase activity than recombinant CYP3A4 expressed alone and supplemented with purified rabbit CYPOR. The spectral P450 content of CYP3A4-OR T.ni microsomes was 107 pmol/mg microsomal protein and the cytochrome c reductase activity was 3904 units/mg. Recombinant CYP3A4-OR was catalytically similar to human liver CYP3A4 based on similarities in the testosterone metabolite profile, time course of metabolite formation, Vmax and Km values (for CYP3A4-OR, Vmax was 8.8 nmol/min/mg microsomal protein [70 nmol/min/nmol CYP3A4] and Km was 33 microM), the extent of inhibition by 100 microM troleandomycin (> 75%) in the presence of 25 microM testosterone, and the degree of P450 activation in the presence of 20 microM 7,8-benzoflavone. The coexpression of recombinant cytochrome b5 with CYP3A4-OR did not result in an additional increase in CYP3A4-OR activity.
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PMID:CYP3A4 expressed by insect cells infected with a recombinant baculovirus containing both CYP3A4 and human NADPH-cytochrome P450 reductase is catalytically similar to human liver microsomal CYP3A4. 777 80

1. The effects of feeding allyl sulphides to rat (2000 ppm of the diet for 15 days) were investigated on various microsomal hepatic drug-metabolizing enzymes by their immunochemical detection and catalytic activity. 2. Allyl sulphides provoked a temporary dietary restriction, which enhanced the microsomal level of P450 and the activities of NADH-cytochrome c reductase and p-hydroxybiphenyl UDP-glucuronyltransferase (UDPGT 2), and lowered the activities of p-nitrophenol hydroxylase (PNPH), N-nitrosodimethylamine demethylase (NDMAD), laurate omega-hydroxylase (LAH) and glutathione S-transferase (GST). Therefore, pair-fed animals were used as a more relevant control for the dietary effects of allyl sulphides. 3. Diallyl sulphide (DAS) as well as diallyl disulphide (DADS) produced an enhancement of the microsomal level of P4501A2, 2B1/2 and 3A1/2, and epoxide hydrolase (EH) proteins, with an increase in the enzymatic activities they catalyse: ethoxyresorufin O-deethylase (EROD), aryl hydrocarbon hydroxylase (AHH), methoxyresorufin O-demethylase (MROD), ethoxycoumarin O-deethylase (ECOD), pentoxyresorufin O-depentylase (PROD), benzoxyresorufin O-debenzylase (BROD) and EH. Although P4502E1 proteins were lowered on treatment, NDMAD activity was not modified, and PNPH activity was even enhanced by allyl sulphides. Only DAS treatment raised erythromycin N-demethylase (ERDM) activity. 4. Both DAS and DADS increased the activity of GST and p-nitrophenol UDP-glucuronyltransferase (UDPGT 1), whereas UDPGT 2 activity was enhanced only by DAS.
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PMID:Modification of hepatic drug-metabolizing enzymes in rat fed naturally occurring allyl sulphides. 801 91

Monkey kidney COS1 cells transiently transfected with plasmids pMT2-cytochrome P450 1A1 (CYP1A1), pMT2-cytochrome P450 reductase (P450 reductase), and pMT2-NAD(P)H:quinone oxidoreductase1 (NQO1 or DT diaphorase), individually or in combination, expressed significantly elevated levels of the respective enzyme(s). The transfected cells were homogenized to break cell membranes without affecting the nuclei and incubated with benzo[a]pyrene (BP) to determine the role of cDNA-encoded enzymes in metabolic activation and/or detoxification of BP. These studies were performed by measuring the capacity of the transfected cells to form DNA adducts as determined by 32P postlabeling and protein adduct detection. Cotransfection of the COS1 cells with cDNAs encoding CYP1A1 and P450 reductase resulted in eight distinct BP-DNA adducts. Inclusion of cDNA encoding NQO1 along with CYP1A1 and P450 reductase in transfection reduced the number of DNA adducts to six. The two lost DNA adducts were specifically eliminated due to the presence of cDNA-derived NQO1 activity. Subsequent experiments with BP-1,6-quinone, BP-3,6-quinone, and BP-6,12-quinone identified these two adducts as those of BP quinones. In an in vitro system, BP-3,6-quinone produced two adducts with deoxyguanosine (dG) but not with dA, dC, and dT. Furthermore, the positions of BP-3,6-quinone-dG adducts on TLC plate correspond to those that are prevented by cDNA-derived NQO1, thus identifying these adducts as BP quinones of dG. In addition, NQO1 reduced the amount of protein-BP adducts generated by CYP1A1 and P450 reductase into transfected COS1 cells. These results show that semiquinones can directly bind to DNA and demonstrate that NQO1 activity can specifically reduce the binding of quinone metabolites of BP generated by CYP1A1 and P450 reductase to DNA and protein.
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PMID:NAD(P)H:quinone oxidoreductase1 (DT diaphorase) specifically prevents the formation of benzo[a]pyrene quinone-DNA adducts generated by cytochrome P4501A1 and P450 reductase. 807 96

Cytochrome P450BM-3 from Bacillus megaterium is a soluble, catalytically self-sufficient fatty acid mono-oxygenase that, in structural organization and amino acid sequence, resembles the Class II (microsomal) P450 systems. Its single polypeptide chain contains both a P450 heme domain and an NADPH:P450 reductase domain, each of which bears significant homology with its microsomal counterparts. We report here the critical nature of three amino acids in the reductase domain of this enzyme with respect to FMN binding and catalytic activity. We used site-directed mutagenesis to change glycine 570 to bulkier amino acids; none of these mutant enzymes contained FMN after purification. We also made substitutions for tryptophan 574 and tyrosine 536, which by sequence analogy (Porter, T. D. (1991) Trends Biochem. Sci. 16, 154-158) were proposed to bind FMN through stacking of the aromatic rings with the isoalloxazine ring of the flavin. Mutants of tryptophan 574 which retained the aromatic side chain contained no less than 0.85 mol of FMN per mol of enzyme, while aspartate and glycine substitutions yielded enzymes which did not incorporate FMN. Substitution of tyrosine 536 with aspartate gave an enzyme which contained 0.44 mol of FMN per mol of enzyme but was inactive as a fatty acid hydroxylase and had only 2% of wild-type cytochrome c reductase activity, while the glycine mutant at this position bound no FMN. Furthermore, although all of the mutant enzymes contained 1 mol of FAD per mol of enzyme, the Y536D mutant and those entirely lacking FMN retained no more than 40% of wild-type ferricyanide reductase activity. By assaying these enzymes in the presence of added FMN, we were able to assess the relative importance of the residues in the wild-type sequence with respect to their contribution to FMN binding. In addition, the aromatic mutants of tryptophan 574, which were nearly as active in cytochrome c reduction as wild-type P450BM-3, were only 20% as active in myristate hydroxylation as the wild-type enzyme, suggesting that this amino acid plays an important role in the flow of electrons between the P450 heme and reductase domains.
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PMID:Critical residues involved in FMN binding and catalytic activity in cytochrome P450BM-3. 846 85

Effects of acute or subchronic administration of human placental extract (HPE), a worldwide clinically used agent, on hepatic drug metabolizing enzyme activities were evaluated in rats. Hepatic microsomal cytochrome P-450 (Cyt. P450) and cytochrome b5 (Cyt. b5) contents and cytosolic glutathione S-transferase (GST) activities were maximally induced after various periods of time following a single intraperitoneal injection of HPE (4 ml/kg) whereas microsomal UDP-glucuronyltransferase (UDPGT) activities were inhibited significantly. All these altered effects were returned almost to the basal levels after 96 h of treatment. Subchronic treatment (30 days) with HPE (1,2 or 4 ml/kg) afforded a significant induction of Cyt. P-450 and Cyt. b5 levels and that of GST activities with a concurrent suppression of the activities of UDPGT and these results were found to be dose-dependent. However, microsomal NADPH cytochrome c reductase activity was not affected either by acute or subchronic treatment. The observed variations in the levels and activities of above house-keeping enzymes were discussed in relation to the possible carcinogenic risk of long-term treatment with this pharmaceutical agent.
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PMID:Effects of human placental extract on hepatic drug metabolizing enzyme. 854 51

Mitomycin C (MMC), an alkylating anti-tumor agent, was activated by non-enzymatic and enzymatic mechanisms leading to DNA binding and adduct formation. However, it was enzymatically, not non-enzymatically, activated MMC which induced inter-strand DNA cross-linking, a major determinant of cell death. The enzymatic activation of MMC was catalyzed by microsomal NADPH:cytochrome P450 reductase (P450 reductase) and cytosolic enzyme activities. Human P450 reductase, transiently expressed from its cDNA in the COSI cells, metabolically activated MMC to generate 9 specific MMC-DNA adducts and induced inter-strand DNA cross-linking. Co-chromatography of the MMC-DNA adducts generated by P450 reductase and sodium borohydride in separate experiments indicated that MMC was metabolized by P450 reductase to produce 2,7-diaminomitosenes that exhibited binding to deoxyguanosine. Several experiments indicated that cytosolic enzymes which catalyzed reductive activation of MMC and DNA cross-linking included NAD(P)H:quinone oxidoreductaseI (NQOI or DT diaphorase) when present in extremely high concentrations and a unique cytosolic activity. The unique cytosolic activity was present in several mammalian cells and mouse colon and liver but absent in mouse kidney. The unique activity had properties of a diaphorase but was distinct from NQOI because of a lack of correlation between NQOI (2,6-dichlorophenolindophenol reduction) activity and the amount of MMC-reductive activation leading to DNA cross-linking. This activity was also distinct from xanthine oxidoreductase and NADH-cytochrome b5 reductase, 2 other enzymes that catalyze metabolic activation of MMC, because the unique activity was not inhibited by allopurinol (an inhibitor of xanthine oxidoreductase) and its activity was the same with NADH and NADPH (cytochrome b5 reductase is specific to NADH).
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PMID:Non-enzymatic and enzymatic activation of mitomycin C: identification of a unique cytosolic activity. 856 27


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