Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:1.8.1.4 (diaphorase)
 2,754 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

From Trypanosoma cruzi, the causative agent of Chagas' disease, a lipoamide dehydrogenase was isolated. The enzyme, an FAD-cystine oxidoreductase, shares many physical and chemical properties with T. cruzi trypanothione reductase, the key enzyme of the parasite's thiol metabolism. 1. From 60 g epimastigotic T. cruzi cells, 2.7 mg lipoamide dehydrogenase was extracted. The flavoenzyme was purified 3000-fold to homogeneity with an overall yield of 26%. 2. The enzyme is a dimer with a subunit Mr of 55,000. With 1 mM lipoamide (Km approximately 5 mM) and 100 microM NADH (Km = 23 microM), the specific activity at pH 7.0 is 297 U/mg. 3. With excess NADH, the enzyme is reduced to the EH2.NADH complex and, by addition of lipoamide, it is reoxidized, indicating that it can cycle between the oxidized state E and the two-electron-reduced state, EH2. 4. As shown by N-terminal sequencing of the enzyme, 21 out of 30 positions are identical with those of pig heart and human liver lipoamide dehydrogenase. The sequenced section comprises the GGGPGG stretch, which represents the binding site for the pyrophosphate moiety of FAD. 5. After reduction of Eox to the two-electron-reduced state, the enzyme is specifically inhibited by the nitrosourea drug 1,3-bis(2-chloroethyl)-1-nitrosourea (Carmustine), presumably by carbamoylation at one of the nascent active-site thiols. 6. Polyclonal rabbit antibodies raised against T. cruzi lipoamide dehydrogenase and trypanothione reductase are specific for the respective enzyme, as shown by immunoblots of the pure proteins and of cell extracts.
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PMID:Purification and characterization of lipoamide dehydrogenase from Trypanosoma cruzi. 226 5

We extended our previous studies of the selectivity and mechanism of action as an enzyme inhibitor of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), an antitumor drug now widely used to inactivate glutathione reductase (GSSG-R) experimentally. In contrast to other enzymes examined so far, lipoamide dehydrogenase (LSSLNH2-D) was, like its genetic relative GSSG-R, also strongly inhibited by BCNU. The drug concentration needed to inactivate GSSG-R and LSSLNH2-D was much smaller than that affecting the least resistant of five other flavoenzymes tested. When oxidized, both GSSG-R and LSSLNH2-D were resistant to BCNU, and to be effective, the drug had to interact directly with enzyme protein reduced by its specific pyridine nucleotide. In intact human erythrocytes, GSSG-R was mostly reduced and LSSLNH2-D activity undetectable. The partial genetic homology of GSSG-R and LSSLNH2-D and their special sensitivity to BCNU provided a unique opportunity to define more exactly the site of drug-enzyme interaction through comparative coenzyme studies combined with direct and reciprocal substrate competition experiments. The results, together with earlier data on the prevention of BCNU inhibition by cysteine, indicate that the nitrosourea achieves its relative selectivity against the two related flavoenzymes by interacting with at least one of the two reduced cysteinyls located within their oxidoredox active site. For GSSG-R, the attacked cysteinyl is most probably Cys-58.
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PMID:Active site-specific inhibition by 1,3-bis(2-chloroethyl)-1-nitrosourea of two genetically homologous flavoenzymes: glutathione reductase and lipoamide dehydrogenase. 392 Mar 38

Human platelets exposed in vitro to increasing amounts of BCNU rapidly develop a progressive, relatively selective, and almost complete deficiency of GSSG-R activity. Several other enzymes are not inhibited when intact platelets are exposed to the nitrosourea; lipoamide dehydrogenase was investigated because of the remarkable similarity of the structure of its active site with that of GSSG-R. BCNU inhibits lipoamide dehydrogenase and GSSG-R only when they are in the reduced state; in the intact platelet, lipoamide dehydrogenase (unlike GSSG-R) is oxidized and is therefore unaffected. This is the first documentation of lipoamide dehydrogenase activity in platelets. After BCNU exposure, there is a reduced release of 14C-serotonin in response to collagen; the cells become incapable of aggregating in response to even large doses of epinephrine, ADP, collagen, or arachidonic acid, with loss of both primary and secondary waves of aggregation. At higher doses of BCNU, there is also a diminished PF-3 activity of intact platelets; sonication of drug-treated platelets normalizes coagulant activity. The drug-induced functional abnormalities occur despite preservation of the number of platelets, their electron microscopic appearance, and their capacity to take up 14C-serotonin. BCNU induced GSSG-R deficiency precedes the development of the earliest evidence of platelet dysfunction, and almost all of the enzyme's activity must be abolished before any functional abnormality becomes detectable. A small fraction of GSSG-R activity is essential for platelet function, and BCNU provides a powerful new tool to investigate the role of the enzymatic reduction of glutathione in platelet physiology and pathology.
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PMID:Glutathione reductase deficiency and platelet dysfunction induced by 1,3-bis(2-chloroethyl)-1-nitrosourea. 668 96

The geno- and cytotoxicity of chromate, an important environmental pollutant, is partly attributed to the flavoenzyme-catalyzed reduction with the concomitant formation of reactive oxygen species. The aim of this work was to characterize the role of NAD(P)H:quinone oxidoreductase (NQO1, DT-diaphorase, EC 1.6.99.2) and glutathione reductase (GR, EC 1.6.4.2) in the mammalian cell cytotoxicity of chromate, which was evidenced controversially so far. The chromate reductase activity of NQO1 was higher than that of GR, but lower than that of lipoamide dehydrogenase (EC 1.6.4.3), ferredoxin:NADP+ reductase (EC 1.18.1.2), and NADPH: cytochrome P-450 reductase (EC 1.6.2.4). The reduction of chromate by NQO1 was accompanied by the formation of reactive oxygen species. The concentration of chromate for 50% survival of bovine leukemia virus-transformed lamb kidney fibroblasts (line FLK) during a 24-h incubation was (22 +/- 4) microM. The cytotoxicity was partly prevented by desferrioxamine, the antioxidant N,N'-diphenyl-p-phenylene diamine and by an inhibitor of NQO1, dicumarol, and potentiated by 1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU), which inactivates GR. The NADPH-dependent chromate reduction by digitonin-permeabilized FLK cells was partly inhibited by dicumarol and not affected by BCNU. Taken together, these data indicate that the oxidative stress-type cytotoxicity of chromate in FLK cells may be partly attributed to its reduction by NQO1, but not by GR. The effect of BCNU on the chromate cytotoxicity may indicate that the general antioxidant action of reduced glutathione is more important than its prooxidant activities arising from the reactions with chromate.
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PMID:Prooxidant cytotoxicity of chromate in mammalian cells: the opposite roles of DT-diaphorase and glutathione reductase. 1729 2