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 objective of this study was to elucidate the mechanisms of mitochondrial H2O2 generation in mouse organs by determining the nature of their differences in substrate utilization, inhibitor sensitivity, and the site specificity affecting H2O2 production. Mitochondria were isolated from heart, brain, and kidney and the rate of H2O2 generation was measured using the FADH-linked substrates succinate and alpha-glycerophosphate as well as the NADH-linked substrates pyruvate/malate, beta-hydroxybutyrate, and glutamate. Respiratory inhibitors, antimycin and rotenone, were added singly and sequentially to each substrate-supported H2O2 generation reaction mixture to determine the mitochondrial site(s) of generation and the optimal condition(s) for maximal rates of generation. Succinate supported the highest rate of mitochondrial H2O2 generation. Moreover, it was the preferred substrate for the heart mitochondria. alpha-Glycerophosphate is a poor substrate for H2O2 generation in heart mitochondria. Inhibitor studies showed that heart mitochondria were the most sensitive and responsive to antimycin, while brain was the most sensitive to rotenone. A surprising finding was that NADH-linked substrate-supported H2O2 generation in kidney mitochondria was not responsive to rotenone. The contribution from each of the three sites (ubiquinone, NADH dehydrogenase, and alpha-glycerophosphate dehydrogenase) of mitochondrial H2O2 generation to the total was both substrate and organ dependent. Results indicate that assay conditions must be considered before comparisons of sites and rates of mitochondrial H2O2 generation among different organs can be made.
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PMID:Substrate and site specificity of hydrogen peroxide generation in mouse mitochondria. 946 28

To establish biochemical and functional relations during thyroid development, the activity of thyroid peroxidase (TPO), nicotinamide adenine dinucleotide phosphate (NADPH)-cytochrome c reductase and monoamine oxidase (MAO) in a particulate fraction and the iodide transport and organification in slices of bovine fetal thyroid were examined throughout gestation. The cytochemical localization of TPO, H2O2 generating sites and MAO was also studied. Fetal glands were grouped in stages I to V according to increasing developmental features; adult tissues were also analyzed. TPO activity in each of the fetal stages was higher than in the adult; a marked increase was observed in stages IV and V. Iodide transport (T/M) was similar in stages I to V and the adult. Iodide organification in fetal thyroids showed a similar pattern to that of TPO activity. When compared with the adult, at midgestation (stages II to III), a lower iodination coexisted with a higher TPO activity. The activity of NADPH-cytochrome c reductase and MAO, two enzymes previously proposed to participate in thyroid H2O2 generation, did not parallel the level of iodide organification. Cells from stages II to V exhibited a positive cytochemical reaction for TPO in the rough endoplasmic reticulum (RER) and the perinuclear cisternae (PC). In stages IV, V, and adult, TPO was occasionally found in apical vesicles and microvilli, whereas H2O2 was detected within the RER and the PC. MAO reaction was positive in adult, but not in fetal thyroid. These results indicate that a high TPO activity accompanied the onset of the organification process during fetal thyroid development. The level of iodination was associated with the presence of TPO at a proper site rather than to the level of TPO activity. Evidence against a role of NADPH-cytochrome c reductase and MAO in the iodide organification was obtained.
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PMID:Biochemical and functional changes during the bovine fetal thyroid development. 949 57

The molecular mechanism of the anthracycline-dependent development of cardiotoxicity is still far from being clear. However, it is generally accepted, that mitochondria play a significant role in triggering this organ specific injury. The results presented in this study demonstrate that, in contrast to liver mitochondria, isolated heart mitochondria shuttle single electrons to adriamycin, giving rise to oxygen radical formation via autoxidation of adriamycin semiquinones. This one electron reduction of anthracyclines is catalyzed by the exogenous NADH dehydrogenase associated with complex I of heart mitochondria, an enzyme which is lacking in liver mitochondria. Upon addition of NADH heart mitochondria generate significant amounts of adriamycin semiquinones while liver mitochondria were ineffective. Adriamycin semiquinones undergo both autoxidation leading to superoxide radical release and complex reactions under formation of adriamycin aglycone. Due to the high lipophilicity adriamycin aglycones accumulate in the inner mitochondrial membrane where they interfere with electron carriers of the respiratory chain. Adriamycin aglycone semiquinones emerging from an interaction with complex I were found to trigger homolytic cleavage of H2O2 which results in the formation of hydroxyl radicals. As demonstrated in this study the activation of adriamycin by the exogenous NADH dehydrogenase of cardiac mitochondria initiates a cascade of reaction steps leading to the establishment of oxidative stress. Our experiments suggest the exogenous NADH dehydrogenase of heart mitochondria to play a key role in the cardiotoxicity of adriamycin. This organ-specific enzyme initiates a sequence of one electron transfer reactions ending up in the establishment of oxidative stress.
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PMID:The exogenous NADH dehydrogenase of heart mitochondria is the key enzyme responsible for selective cardiotoxicity of anthracyclines. 961 42

Oxidants are important in the regulation of signal transduction and gene expression. Multiple classes of genes are transcriptionally activated by oxidants and are implicated in different phenotypic responses. In the present study, we performed differential mRNA display to elucidate genes that are induced or repressed after exposure of rat lung epithelial (RLE) cells to H2O2 or crocidolite asbestos, a pathogenic mineral that generates oxidants. After 8 or 24 hr of exposure, RNA was extracted, reverse transcribed, and amplified by polymerase chain reaction with degenerate primers to visualize alterations in gene expression. The seven clones obtained were sequenced and encoded the mitochondrial genes, NADH dehydrogenase subunits ND5 and ND6, and 16S ribosomal RNA. Evaluation of their expression by Northern blot analysis revealed increased expression of 16S rRNA after 1 or 2 hr of exposure to H2O2. At later time periods (4 and 24 hr), mRNA levels of 16S rRNA and NADH dehydrogenase were decreased in H2O2-treated RLE cells when compared to sham controls. Crocidolite asbestos caused increases in 16S rRNA levels after 8 hr of exposure, whereas after 24 hr of exposure to asbestos, 16S rRNA levels were decreased in comparison to sham controls. In addition to these oxidants, the nitric oxide generator spermine NONOate caused similar decreases in NADH dehydrogenase mRNA levels after 4 hr of exposure. The present data and previous studies demonstrated that all oxidants examined resulted in apoptosis in RLE cells during the time frame where alterations of mitochondrial gene expression were observed. As the mitochondrion is a major organelle that controls apoptosis, alterations in expression of mitochondrial genes may be involved in the regulation of apoptosis.
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PMID:Modulation of mitochondrial gene expression in pulmonary epithelial cells exposed to oxidants. 978 97

The participation of oxidative mechanisms in major histocompatibility complex (MHC) class II-restricted antigen presentation was studied in vitro. In general, antigen processing is inhibited when peritoneal macrophages (MO) are incubated with scavengers of reactive oxygen intermediates (ROI): mannitol (an.OH scavenger), dimethylurea (DMTU, which reacts with H2O2 and HOCl) and NCO-700 (an epoxysuccinic acid derivative which inhibits oxidant production by activated phagocytes and can scavenge reactive oxygen species in both NaOCl and hypoxanthine (XOD) systems). However, neither rotenone and antimycins (inhibitors of O-2 production at the NADH dehydrogenase and ubiquinone-cytochrome b regions, respectively) nor aminoguanidine (an inducible nitric oxide synthase inhibitor) impaired antigen presentation, thus indirectly discarding the participation of mitochondrial oxidation and reactive nitrogen intermediates (RNI) in antigen processing. ROI scavengers do not inhibit the MHC class II-restricted presentation of antigens that need processing but have their disulphide bonds reduced. It can be shown that oxidation of protein antigens (either by chlorination or performic acid treatment) allow protein unfolding and enhance both processing and exposure of immunogenic epitopes to specific T cells.
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PMID:Oxidation of defined antigens allows protein unfolding and increases both proteolytic processing and exposes peptide epitopes which are recognized by specific T cells. 982 92

2-Amino-3-carboxy-1,4-naphthoquinone, discovered as a novel bifidogenetic growth stimulator (BGS), has been characterized by determination of redox and acid-base equilibria, partition properties, and UV-vis and electron spin resonance spectral properties. BGS is proposed to function as an electron transfer mediator from NADH to O2. BGS is reduced by NADH-reduced diaphorase (or related enzymes) and the reduced BGS is reoxidized by autoxidation and a peroxidase-catalyzed reaction. The proposed reaction would spare pyruvate as an important metabolic intermediate, and minimize the cytotoxic effects of H2O2 generated by the autoxidation. Kinetic studies were performed in model enzymatic systems using 2-methyl-1,4-naphthoquinone (VK3) as a reference compound with a very weak growth-stimulating effect. The results support our proposal and reveal the superiority of BGS to VK3 as an electron transfer mediator in the proposed reactions.
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PMID:Mechanistic study on the roles of a bifidogenetic growth stimulator based on physicochemical characterization. 983 15

P. S. Alban et al. (J. Appl. Microbiol. (1998) 85, 875-882) reported that a mutant H2O2-resistant strain of Spirullum (S.) volutans showed constitutive overexpression of a protein whose amino acid sequence and molecular weight closely resembled that of a subunit of rubrerythrin, a non-heme iron protein with no known function. They also reported that the mutant strain, but not the wild-type, showed NADH peroxidase activity. Here we demonstrate that rubrerythrin and nigerythrin from Desulfovibrio vulgaris and rubrerythrin from Clostridium perfringens show NADH peroxidase activities in an in vitro system containing NADH, hydrogen peroxide, and a bacterial NADH oxidoreductase. The peroxidase specific activities of the rubrerythrins with the "classical" heme peroxidase substrate, o-dianisidine, are many orders of magnitude lower than that of horseradish peroxidase. These results are consistent with the phenotype of the H2O2-resistant strain of S. volutans. The reaction of reduced (i.e., all-ferrous) rubrerythrin with excess O2 takes several minutes, whereas the anaerobic reaction of reduced rubrerythrin with hydrogen peroxide is on the millisecond time scale and results in full oxidation of all iron centers to their ferric states. Rubrerythrins could, thus, function as the terminal components of NADH peroxidases in air-sensitive bacteria and archaea.
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PMID:NADH peroxidase activity of rubrerythrin. 1004 6

The fitness of organisms depends upon the rate at which they generate superoxide (O-2) and hydrogen peroxide (H2O2) as toxic by-products of aerobic metabolism. In Escherichia coli these oxidants arise primarily from the autoxidation of components of its respiratory chain. Inverted vesicles that were incubated with NADH generated O-2 and H2O2 at accelerated rates either when treated with cyanide or when devoid of quinones, implicating an NADH dehydrogenase as their source. Null mutations in the gene encoding NADH dehydrogenase II averted autoxidation of vesicles, and its overproduction accelerated it. Thus NADH dehydrogenase II but not NADH dehydrogenase I, respiratory quinones, or cytochrome oxidases formed substantial O-2 and H2O2. NADH dehydrogenase II that was purified from both wild-type and quinone-deficient cells generated approximately 130 H2O2 and 15 O-2 min-1 by autoxidation of its reduced FAD cofactor. Sulfite reductase is a second autoxidizable electron transport chain of E. coli, containing FAD, FMN, [4Fe-4S], and siroheme moieties. Purified flavoprotein that contained only the FAD and FMN cofactors had about the same oxidation turnover number as did the holoenzyme, 7 min-1 FAD-1. Oxidase activity was largely lost upon FMN removal. Thus the autoxidation of sulfite reductase, like that of the respiratory chain, occurs primarily by autoxidation of an exposed flavin cofactor. Great variability in the oxidation turnover numbers of these and other flavoproteins suggests that endogenous oxidants will be predominantly formed by only a few oxidizable enzymes. Thus the degree of oxidative stress in a cell may depend upon the titer of such enzymes and accordingly may vary with growth conditions and among different cell types. Furthermore, the chemical nature of these reactions was manifested by their acceleration at high temperatures and oxygen concentrations. Thus these environmental parameters may also directly affect the O-2 and H2O2 loads that organisms must bear.
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PMID:The identification of primary sites of superoxide and hydrogen peroxide formation in the aerobic respiratory chain and sulfite reductase complex of Escherichia coli. 1018 94

Dihydrolipoamide dehydrogenase (LADH) lipoamide reductase activity decreased whereas enzyme diaphorase activity increased after LADH treatment with myeloperoxidase (MPO) dependent systems (MPO/H2O2/halide, MPO/NADH/halide and MPO/H2O2/nitrite systems. LADH inactivation was a function of the composition of the inactivating system and the incubation time. Chloride, iodide, bromide, and the thiocyanate anions were effective complements of the MPO/H2O2 system. NaOCl inactivated LADH, thus supporting hypochlorous acid (HOCl) as putative agent of the MPO/H2O2/NaCl system. NaOCl and the MPO/H2O2/NaCl system oxidized LADH thiols and NaOCl also oxidized LADH methionine and tyrosine residues. LADH inactivation by the MPO/NADH/halide systems was prevented by catalase and enhanced by superoxide dismutase, in close agreement with H2O2 production by the LADH/NADH system. Similar effects were obtained with lactoperoxidase and horse-radish peroxidase supplemented systems. L-cysteine, N-acetylcysteine, penicillamine, N-(2-mercaptopropionylglycine), Captopril and taurine protected LADH against MPO systems and NaOCl. The effect of the MPO/H2O2/NaNO2 system was prevented by MPO inhibitors (sodium azide, isoniazid, salicylhydroxamic acid) and also by L-cysteine, L-methionine, L-tryptophan, L-tyrosine, L-histidine and reduced glutathione. The summarized observations support the hypothesis that peroxidase-generated "reactive species" oxidize essential thiol groups at LADH catalytic site.
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PMID:Inactivation of myocardial dihydrolipoamide dehydrogenase by myeloperoxidase systems: effect of halides, nitrite and thiol compounds. 1019 78

The activities and mRNA abundances of enzymes that regulate the rate of electron flow through the electron transport chain (ETC), including NADH dehydrogenase, succinate dehydrogenase, and cytochrome c oxidase, were examined in young and senescent fetal lung fibroblasts (WI-38). We also determined the activities and mRNA abundances of antioxidant defenses including superoxide dismutase, catalase, and glutathione peroxidase. We confirmed our previous report of a senescence-related increase in the abundance of ND4, a mitochondrially encoded subunit of NADH dehydrogenase. The activities of cytochrome c oxidase and NADH dehydrogenase were also elevated in senescent cultures. No differences were observed in the mRNA abundances of COX-1, a mitochondrially encoded subunit of cytochrome c oxidase or of nuclearly encoded subunits of various electron transport components (SD, COX-4, and ND 51). Lucigenin-detected chemiluminescence and H2O2 generation were both elevated in senescent cells. Catalase activity was also elevated in senescent fibroblasts. However, no differences in catalase mRNA abundance were observed. A small decrease in GSH peroxidase (GPx) mRNA abundance was observed in senescent cells. No other changes in the activities or mRNA abundances of any of the antioxidant defenses were observed in early and late passage cultures. The relationships between oxidant generation, mitochondrial enzyme activities, and antioxidant defense observed during proliferative senescence are dissimilar to those detected between fetal and postnatal fibroblasts as well as those found between fibroblast lines obtained from young and old individuals. The relevance of the differences between these models is discussed.
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PMID:Differences in electron transport potential, antioxidant defenses, and oxidant generation in young and senescent fetal lung fibroblasts (WI-38). 1036 24


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