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)

The results of the present experiment are shown in terms of the transport of protoheme from mitochondria to apocytochrome b5 when fresh rat liver mitochondria, apocytochrome b5, and cytosol were incubated. The heme transfer protein was purified from rat liver cytosol up to approximately 133-140-fold with a 43% yield by the procedure discussed herein, including Sephadex G-75 and CM-cellulose column chromatography. The final preparation showed apparent homogeneity upon polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Its native form was found to be a dimeric protein with a Mr = 45,000 which consists of a subunit with a Mr = 23,000. In the transporting system, the heme transfer depended on the concentration of mitochondria (donor), apocytochrome b5 (acceptor), and purified transfer protein, respectively. Omission of one of these components led to an almost complete loss of the transfer activity. The transport of mitochondrial protoheme was a rapid reaction which showed approximate linearity until 1.5 min and after that it became saturated. When the functional capacity was tested by the NADH-cytochrome c reductase system, the reconstituted cytochrome b5 expressed its complete original catalytic properties, as well as its characteristic absorption spectra for the hemoprotein. Furthermore, the detailed physicochemical and immunological characterization of the transfer protein provided evidence that the protein is identical with soluble glutathione S-transferase, which conjugates glutathione with a variety of electrophilic compounds. At least one of the glutathione S-transferase isozymes observed was identified as GST-C2, which comprises the subunit of Yb'Yb' by the immunoprecipitation reaction using various anti-glutathione S-transferase isozyme antibodies.
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PMID:Purification and characterization of cytosolic liver protein facilitating heme transport into apocytochrome b5 from mitochondria. Evidence for identifying the heme transfer protein as belonging to a group of glutathione S-transferases. 392 64

The cDNA encoding QPc-9.5 kDa (subunit VII) of bovine heart mitochondrial ubiquinol-cytochrome c reductase was cloned and sequenced. This cDNA is 665 base pairs long with an open reading frame of 246 base pairs that encodes an 81-amino acid mature QPc-9.5 kDa. The insert contains 395 base pairs of a 3'-noncoding sequence with a poly(A) tail. The amino acid sequence of QPc-9.5 kDa deduced from this nucleotide sequence is the same as that obtained by protein sequencing except that residue 61 is tryptophan instead of cysteine. The QPc-9.5 kDa was overexpressed in Escherichia coli JM109 cells as a glutathione S-transferase fusion protein (GST-QPc) using the expression vector, pGEX/QPc. The yield of soluble active recombinant GST-QPc fusion protein depends on the induction growth time, temperature, and medium. Maximum yield of recombinant fusion protein was obtained from cells harvested 3 h postinduction of growth at 27 degrees C on LB medium containing betaine and sorbitol. QPc-9.5 kDa was released from the fusion protein by proteolytic cleavage with thrombin. Isolated recombinant QPc-9.5 kDa showed one protein band in SDS-polyacrylamide gel electrophroesis corresponding to subunit VII of mitochondrial ubiquinol-cytochrome c reductase. Although the isolated recombinant QPc-9.5 kDa is soluble in aqueous solution, it is in a highly aggregated form, with an apparent molecular mass of over 1 million. Addition of detergent deaggreates the isolated protein to the monomeric state, suggesting that the recombinant protein exists as a hydrophobic aggregation in aqueous solution. The recombinant QPc-9.5 kDa binds ubiquinone and shows a spectral blue shift. Upon titration of the recombinant protein with ubiquinone, a saturation behavior is observed, suggesting that the binding is specific and that the recombinant protein may be in the functionally active state.
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PMID:Cloning, gene sequencing, and expression of the small molecular mass ubiquinone-binding protein of mitochondrial ubiquinol-cytochrome c reductase. 759 38

Female F344 rats received an i.p. injection of iron-dextran (600 mg Fe/kg) and then after 1 week were fed a diet containing 0.02% hexachlorobenzene (HCB) for up to 65 weeks. All rats (8/8) which received HCB after iron overload developed multiple hepatic nodules whereas only 3/8 rats administered HCB alone had nodules (average of one per positive liver). These hyperplastic regions were depleted of iron and were often positive for gamma-glutamyl transpeptidase (GGT) and glutathione S-transferase P (GST-P). Telangiectasis and peliosis were prominent features in the lesions. Short-term experiments (5-15 weeks of iron/HCB treatments) showed that GGT and GST-P were induced early in the neoplastic process but not in discrete focal areas. Iron alone also caused some induction of these enzymes. Some cells with induced GST-P in either short or long term experiments also stained positively for this enzyme in the nucleus. Studies of cytochrome P450 mediated activities showed that at 5 and 15 weeks HCB had induced EROD (an estimate of CYP1A1), PROD (CYP2B1 activity) and BROD activities (CYP2B1 but also other isoenzymes). Under the influence of iron overload EROD was significantly depressed from HCB alone, but not the others or cytochrome P450 reductase. Cytosolic glutathione S-transferase activities were also induced by HCB, but, unlike microsomal EROD, preloading with iron enhanced the effects. In contrast, although cytosolic diaphorase activity was induced by HCB, this response was depressed in combination with iron. Glutathione peroxidase (with H2O2 as substrate) was depressed by both iron and HCB. Clearly, iron overload potentiates the neoplastic process induced by HCB in rats, with both enhancing and depressing effects on various enzyme activities induced by this chemical.
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PMID:Enhancement by iron of hepatic neoplasia in rats caused by hexachlorobenzene. 833 Mar 54

Neuronal NO synthase (nNOS) was discovered recently to interact specifically with the protein PIN (protein inhibitor of nNOS) [Jaffrey, S.R. and Snyder, S.H. (1996) Science 274, 774-777]. We have studied the effects on pure NOS enzymes of the same GST-tagged PIN used in the original paper. Unexpectedly, all NOS isoenzymes were inhibited. The IC50 for nNOS was 18 +/- 6 microM GST-PIN with 63 nM nNOS after 30 min at 37 degrees C. Uncoupled NADPH oxidation was inhibited similarly, whereas cytochrome c reductase activity, the K(M) for L-arginine, and dimerization were unaffected. We reconsider the physiological role of PIN in the light of these results.
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PMID:The protein inhibitor of neuronal nitric oxide synthase (PIN): characterization of its action on pure nitric oxide synthases. 968 79

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

o-Quinones are easily formed by oxidation of physiologically relevant catechols. These reactions mainly occur in two specialized cells, catecholaminergic neurons and melanocytes. Both types of cells are related ontogenetically, as they arise from the neural crest during the developmental differentiation. o-Quinones are used to form melanin, a protective pigment formed by different mechanisms in melanocytes and catecholaminergic neurons. However, the reactivity of these quinones makes their presence in the cytosol dangerous for the cell survival and these compounds have been proposed as degenerative and apoptotic agents. Thus, melanin-producing cells show several potential mechanisms to protect themselves against the noxious effects of o-quinones. In melanocytes, the most effective autoprotecting mechanisms are the existence of malanosomes as a confined site for melano-synthesis and the action of tyrosinase-related protein 2 (TRP2) to drive L-dopachrome to 5,6-dihydroxyindole-2-carboxylic acid minimizing the formation of 5,6-dihydroxyindole. In catecholaminergic neurons, recent data suggest that glutathione transferase (GST M2-2 isoenzyme) and macrophage migration inhibitory factor (MIF) are very effective in preventing long-lived formation of dopaminechrome and noradrenochrome, although the detoxification reactions are different (conjugation to GSH or isomerization respectively). These mechanisms are less efficient for adrenochrome, although MIF and GST M1-1 could also catalyze similar reactions using this compound as substrate. In addition, the formation of adrenochrome is still under discussion, and adrenolutin formation could contribute to deactivate its harmful effects. The contribution of D-dopachrome tautomerase to these mechanisms is yet unknown, although in contrast to MIF, that enzyme does not recognize catecholaminechromes as substrates. Diaphorase could also be protective against quinones, since this enzyme catalyzes their bielectronic reduction back to catechols, thus preventing the formation of chrome species. This activity has been described in melanocytes and neurons, so that its contribution should be further investigated. In contrast to diaphorase, cytochrome P450 reductase should not be considered a protective enzyme, since its monoelectronic reduction of quinones leads to formation of semiquinones, that is, even more noxious than the quinones.
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PMID:Neurotoxicity due to o-quinones: neuromelanin formation and possible mechanisms for o-quinone detoxification. 1283 99

Disruption of mitochondria and free radical mediated tissue injury have been reported during cardiotoxicity induced by isoproterenol (ISO), a beta-adrenergic catecholamine. The present study was designed to investigate the effect of the combination of ferulic acid (FA) and ascorbic acid (AA) on the mitochondrial damage in ISO induced cardiotoxicity. Induction of rats with ISO (150 mg/kg b.wt., i.p.) for 2 days resulted in a significant decrease in the activities of respiratory chain enzymes (NADH dehydrogenase and cytochrome c-oxidase), tricarboxylic acid cycle enzymes (isocitrate dehydrogenase, succinate dehydrogenase, malate dehydrogenase, alpha-ketoglutarate dehydrogenase), mitochondrial antioxidants (GPx, GST, SOD, CAT, GSH), cytochromes (b, c, c1, aa3) and in the level of mitochondrial phospholipids. A marked elevation in mitochondrial lipid peroxidation, mitochondrial levels of cholesterol, triglycerides and free fatty acids were also observed in ISO intoxicated rats. Pre-co-treatment with the combination of FA (20 mg/kg b.wt.) and AA (80 mg/kg b.wt.) orally for 6 days significantly enhanced the attenuation of these functional abnormalities and restored normal mitochondrial function when compared to individual drug treated groups. Mitigation of ISO induced biochemical and morphological changes in mitochondria were more pronounced with a combination of FA and AA rather than the individual drug treated groups. Transmission electron microscopic observations also correlated with these biochemical parameters. Hence, these findings demonstrate the synergistic ameliorative potential of FA and AA on mitochondrial function during beta-adrenergic catecholamine induced cardiotoxicity and associated oxidative stress in rats.
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PMID:Ferulic acid with ascorbic acid synergistically extenuates the mitochondrial dysfunction during beta-adrenergic catecholamine induced cardiotoxicity in rats. 1676 44

In this study, zebra mussels, D. polymorpha, were exposed to extracts of sediments obtained from two sites, a contaminated lake (Ketelmeer, Km) and a relatively clean lake (Drontenmeer, Dm). The main objective of this work was to investigate whether six selected biomarkers could discriminate between the two sediments. The selected biomarkers included phase I enzymes such as DT-diaphorase, NADPH-cytochrome c reductase, NADH-cytochrome c reductase, a phase II enzyme (glutathione S-transferase, GST), an antioxidant enzyme, catalase, and the total glutathione, reduced (GSH) and oxidized (GSSG). After a short (24 h) and a long-term (7 days) exposure, the levels of these biomarkers were measured in gills and the rest of soft mussel tissues (soft mussel tissue minus gills) and compared with control values. A decrease of GST level by 20% (P = 0.004) and a 4-fold decrease of total glutathione concentration relative to the control, were observed in the gills of mussels exposed to the more contaminated Km extract. No significant differences in the GST activities were observed in the gills of control and Dm extract-treated mussels (P = 0.23). Although the levels of catalase and NADH-cytochrome c reductase were, in the short-term exposure, unaffected, both activities were, in the long-term exposure, reduced in the gills of the mussels exposed to the contaminated Km extract, compared with control values, by 43% and 20%, respectively. The activities of DT-diaphorase and NADPH-cytochrome c reductase remained unaffected in all exposure conditions. However, the level of NADPH-cytochrome c reductase was found higher in gills than in the rest of soft mussel tissues. This difference in the ratio of the two reductases between the two tissues could account for the observed differential responses of the biomarkers.
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PMID:Differential responses of biomarkers in tissues of a freshwater mussel, Dreissena polymorpha, to the exposure of sediment extracts with different levels of contamination. 1718 75

The aim of the present study was to investigate the interaction of 2,4,6-trinitrotoluene (TNT) with liver biotransformation enzymes in European eel Anguilla anguilla (Linnaeus, 1758). Eels were exposed to 0.5, 1 and 2.5mg/l nominal concentrations of TNT for 6 and 24h. Modulation of CYP1A1, UDPGT and GST genes was investigated by real-time PCR. Total CYP450 content, NADPH cytochrome c reductase activity, CYP1A and CYP2B-like activities, such as EROD, MROD and BROD, as well as GST and UDPGT activities, were measured by biochemical assays. An in vitro study was performed on EROD in order to evaluate catalytic modulation by TNT. No modulation of the CYP1A1 gene or protein was observed in TNT-exposed eels. On the other hand, a significant decline of EROD and MROD activities was observed in vivo. An increase in NADPH cyt c reductase, and phase II enzymes (UDPGT and GST) were observed at both gene expression and activity levels. The overall results indicated that TNT is a potential competitive inhibitor of CYP1A activities. A TNT metabolic pathway involving NADPH cyt c reductase and phase II enzymes is also suggested.
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PMID:Interactions of 2,4,6-trinitrotoluene (TNT) with xenobiotic biotransformation system in European eel Anguilla anguilla (Linnaeus, 1758). 1840 54

Oxidative stress can play a key role in myocardial necrosis. The present study was designed to investigate the effect of alpha-mangostin (an antioxidant phytonutrient) on mitochondrial dysfunction and endothelial nitric oxide synthase (eNOS) expression during isoproterenol-induced myocardial necrosis in rats. Induction of rats with isoproterenol (ISO) (150 mg/kg body weight, intraperitoneally) for 2 days resulted in a significant decrease in the activities of respiratory chain enzymes (NADH dehydrogenase and cytochrome c oxidase), tricarboxylic acid cycle enzymes (isocitrate dehydrogenase, succinate dehydrogenase, malate dehydrogenase, and alpha-ketoglutarate dehydrogenase), mitochondrial antioxidants (GPx, GST, SOD, CAT, and GSH), mitochondrial cytochromes (b, c, c1, and aa3), and adenosine triphosphate level. A marked elevation in mitochondrial lipid peroxidation was also observed in ISO-intoxicated rats. Pretreatment with alpha-mangostin (200 mg/kg body weight) orally for 8 days significantly attenuated these functional abnormalities and restored normal mitochondrial function, when compared to the ISO-intoxicated group of rats. Cardiac eNOS expression was assessed by Western blot. Cardiac eNOS expression and NO level were significantly suppressed in ISO-intoxicated rats. Pretreatment with alpha-mangostin extenuated ISO-induced diminution of eNOS expression and NO level. Transmission electron microscopic observations also correlated with these biochemical parameters. Hence, these findings conclude the ameliorative potential of alpha-mangostin against ISO-induced biochemical and morphological changes in mitochondria, which might be mediated through the NO pathway and by its ability at quenching free radicals.
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PMID:Mitigation of mitochondrial dysfunction and regulation of eNOS expression during experimental myocardial necrosis by alpha-mangostin, a xanthonic derivative from Garcinia mangostana. 1979 27


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