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)

1. We have isolated a cDNA encoding the 24 kDa subunit, an iron-sulfur protein, of mitochondrial NADH dehydrogenase from a human fibroblast cDNA library by colony hybridization using a rat 24 kDa subunit cDNA as a probe. 2. The presequence predicted from the human cDNA sequence is typical of precursors to mitochondrial proteins in a high content of basic residues and in the absence of acidic ones. 3. The mature form of the human 24 kDa subunit shows 95% homology with its rat counterpart. Five cysteine residues are conserved among human, rat and bovine; four of these are expected to be involved in the binding of a binuclear iron-sulfur cluster.
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PMID:Cloning and sequencing of a cDNA encoding the precursor to the 24 kDa iron-sulfur protein of human mitochondrial NADH dehydrogenase. 258 50

Dietary iron deficiency in rats results in increased blood glucose turnover and recycling. We measured the rates of glucose production in isolated hepatocytes from iron-sufficient (Fe+) and iron-deficient (Fe-) rats to assess the intrinsic capacity of the Fe- liver to carry out gluconeogenesis. Low-iron and control diets were given to 21-day-old female rats. After 4-5 wk, hemoglobin concentrations averaged 4.1 g/dl in the Fe- and 14.3 g/dl in the Fe+ animals. In the hepatocytes from Fe- rats, there was a 35% decrease in the rate of glucose production from 1 mM pyruvate + 10 mM lactate, a 48% decrease from 0.1 mM pyruvate + 1 mM lactate, a 39% decrease from 1 mM alanine, and a 48% decrease from 1 mM glycerol. The addition of 5 microM norepinephrine or 0.5 microM glucagon to the incubation media produced stimulatory effects on hepatocytes from both Fe- and Fe+ rats, resulting in the maintenance of an average difference of 38% in the rates of gluconeogenesis between the two groups. Studies on isolated liver mitochondria and cytosol revealed alpha-glycerophosphate-cytochrome c reductase and phospho(enol)pyruvate carboxykinase activities to be decreased by 27% in Fe- rats. We conclude that because severe dietary iron deficiency decreases gluconeogenesis in isolated rat hepatocytes, the increased gluconeogenesis demonstrated by Fe- rats in vivo is attributable to increased availability of gluconeogenic substrates and upregulation of the pathway.
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PMID:Iron deficiency decreases gluconeogenesis in isolated rat hepatocytes. 260 20

The quinonoid anthracycline, doxorubicin (Adriamycin) is a potent anti-neoplastic agent whose clinical use is limited by severe cardiotoxicity. Mitochondrial damage is a major component of this cardiotoxicity, and rival oxidative and non-oxidative mechanisms for inactivation of the electron transport chain have been proposed. Using bovine heart submitochondrial preparations (SMP) we have now found that both oxidative and non-oxidative mechanisms occur in vitro, depending solely on the concentration of doxorubicin employed. Redox cycling of doxorubicin by Complex I of the respiratory chain (which generates doxorubicin semiquinone radicals, O2-, H2O2, and .OH) caused a 70% decrease in the Vmax. for NADH dehydrogenase during 15 min incubation of SMP, and an 80% decrease in NADH oxidase activity after 2 h incubation. This inactivation required only 25-50 microM-doxorubicin and represents true oxidative damage, since both NADH (for doxorubicin redox cycling) and oxygen were obligatory participants. The damage appears localized between the NADH dehydrogenase flavin (site of doxorubicin reduction) and iron-sulphur centre N-1. Succinate dehydrogenase, succinate oxidase, and cytochrome c oxidase activities were strongly inhibited by higher doxorubicin concentrations, but this phenomenon did not involve doxorubicin redox cycling (no NADH or oxygen requirement). Doxorubicin concentrations of 0.5 mM were required for 50% decreases in these activities, except for cytochrome c oxidase which was only 30% inhibited following incubation with even 1.0 mM-doxorubicin. Our results indicate that low concentrations of doxorubicin (50 microM or less) can catalyse a site-specific oxidative damage to the NADH oxidation pathway. In contrast, ten-fold higher doxorubicin concentrations (or more) are required for non-oxidative inactivation of the electron transport chain; probably via binding to cardiolipin and/or generalized membrane chaotropic effects. The development of agents to block doxorubicin toxicity in vivo will clearly require detailed clinical studies of doxorubicin uptake in the heart.
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PMID:Oxidative and non-oxidative mechanisms in the inactivation of cardiac mitochondrial electron transport chain components by doxorubicin. 271 42

An investigation into the biogenesis of several of the nuclear-encoded subunits of the iron-protein fragment of mitochondrial NADH dehydrogenase was undertaken utilising a bovine kidney cell line (NBL-1). Inhibition of import was achieved by treating the cells with the uncoupler carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP) and it was demonstrated that the 75-kDa, 51-kDa and 49-kDa components of the enzyme were synthesised as larger polypeptides of 76-kDa, 52-kDa and 53-kDa, respectively. The precursors could subsequently be processed to the mature subunits by reversing the FCCP treatment and chasing for 45 min at 37 degrees C. Subcellular localisation studies using the detergent digitonin illustrated that the 76-kDa, 52-kDa and 53-kDa precursor forms were almost exclusively located in the soluble fraction of the cell, whereas the mature and pulse-chased proteins fractionated with the particulate portion of the cell. Although the mature 30-kDa and 24-kDa subunits of NADH dehydrogenase could be visualised, their precursor forms went undetected in this system.
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PMID:Biosynthetic studies of several of the nuclear-encoded subunits of mammalian NADH dehydrogenase. 273 8

The production of potent oxygen radicals by microsomal reaction systems has been well characterized. Relatively little attention has been paid to generation of oxygen radicals by liver nuclei, or to the interaction of nuclei with different ferric complexes to catalyze NADH- or NADPH-dependent production of reactive oxygen intermediates. Intact rat liver nuclei were capable of catalyzing an iron-dependent production of .OH as reflected by the oxidation of .OH scavenging agents such as 2-keto-4-thiomethylbutyrate, dimethyl sulfoxide, and t-butyl alcohol. Inhibition of .OH production by catalase implicates H2O2 as the precursor of .OH generated by the nuclei, whereas superoxide dismutase had only a partially inhibitory effect. The production of .OH with either cofactor was striking increased by addition of ferric-EDTA or ferric-diethylenetriamine-pentaacetic acid (DTPA) whereas ferric-ATP and ferric-citrate were not effective catalysts. All these ferric complexes were reduced by the nuclei in the presence of either NADPH or NADH. The pattern of iron chelate effectiveness in catalyzing lipid peroxidation by nuclei was opposite to that of .OH production; with either NADH or NADPH, nuclear lipid peroxidation was increased by the addition of ferric ammonium sulfate, ferric-ATP, or ferric-citrate, but not by ferric-EDTA or ferric-DTPA. NADPH-dependent nuclear lipid peroxidation was insensitive to catalase, superoxide dismutase, or .OH scavengers; the NADH-dependent reaction showed a partial sensitivity (30 to 40%) to these additions. The overall patterns of .OH production and lipid peroxidation by the nuclei are similar to those shown by microsomes, e.g., effect of ferric complexes, sensitivity to antioxidants; however, rates with the nuclei are less than 20% those of microsomes, which reflect the lower activities of NADPH- and NADH-cytochrome c reductase in the nuclei. The potential for nuclei to reduce ferric complexes and catalyze production of .OH-like species may play a role in the susceptibility of the genetic material to oxidative damage under certain conditions since such radicals would be produced site-directed and not exposed to cellular antioxidants.
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PMID:Interaction of ferric complexes with rat liver nuclei to catalyze NADH-and NADPH-Dependent production of oxygen radicals. 277 54

Considerable evidence suggests that the release of iron from ferritin is a reductive process. A role in this process has been proposed for two hepatic enzymes, namely xanthine oxidoreductase and an NADH oxidoreductase. The abilities of xanthine and NADH to serve as a source of reducing power for the enzyme-mediated release of ferritin iron (ferrireductase activity) were compared with turkey liver and rat liver homogenates. The maximal velocity (Vmax.) for the reaction with NADH was 50 times greater than with xanthine; however, the substrate concentration required to achieve half-maximal velocity (Km) was 1000 times less with xanthine than with NADH. NADPH could be substituted for NADH with little loss in activity. Dicoumarol did not inhibit the reaction with NADH or NADPH, demonstrating that the ferrireductase activity with those substrates was not the result of the liver enzyme 'DT-diaphorase' [NAD(P)H dehydrogenase (quinone)]. A flavin nucleotide was required for ferrireductase activity with rat and turkey liver cytosol when xanthine, NADH or NADPH was used as the reducing substrate. FMN yielded twice the activity with NADH or NADPH, whereas FAD was twice as effective with xanthine as substrate. Kinetic comparisons, differences in lability and partial chromatographic resolution of the ferrireductase activities with the two types of reducing substrates strongly indicate that the ferrireductase activities with xanthine and NADH are catalysed by separate enzyme systems contained in liver cytosol. Complete inhibition by allopurinol of the ferrireductase activity endogenous to undialysed liver cytosol preparations and the ability of xanthine to restore equivalent activity to dialysed preparations indicate that the source of reducing power for the endogenous activity is xanthine. These studies suggest that xanthine, NADH or NADPH can serve as a source of reducing power for the enzyme-mediated reduction of ferritin iron, with a flavin nucleotide serving as the shuttle of electrons from the enzymes to the ferritin iron.
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PMID:The mobilization of ferritin iron by liver cytosol. A comparison of xanthine and NADH as reducing substrates. 277 99

The inhibition of NADH dehydrogenase by 1-methyl-4-phenylpyridinium (MPP+) leading to ATP depletion has been proposed to explain cell death in the expression of the neurotoxicity of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Electron paramagnetic resonance studies show no effect of MPP+ on the reduction of the iron-sulfur clusters of NADH dehydrogenase. Mitochondria inhibited by MPP+ were sonicated and both the NADH oxidase and the NADH-Q reductase activities were measured. NADH oxidase activity was not fully restored to control levels, but NADH-Q reductase activity was the same as that of the control. Neither succinate-oxidase nor succinate-Q reductase activities were inhibited. These data indicate that MPP+ interaction with NADH dehydrogenase interferes with the passage of electrons from the iron-sulfur cluster of highest potential to endogenous Q10 but that the inhibition can be relieved by the addition of a small, water-soluble Q analog. Inhibition at this site is sufficient to explain the inhibition of respiration and no inhibition of other mitochondrial functions was observed.
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PMID:The inhibition site of MPP+, the neurotoxic bioactivation product of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine is near the Q-binding site of NADH dehydrogenase. 282 83

Coenzyme QH2-cytochrome c reductase is a multisubunit complex of the mitochondrial respiratory chain. Mutants of Saccharomyces cerevisiae with lesions in cytochromes b, c1, the non-heme iron protein, and the noncatalytic subunits have been used to study several aspects of the assembly of the complex. Strains with mutations in single subunits exhibit a variety of different phenotypes. Mutants in the 17-kDa (core 3) subunit grow normally on a nonfermentable substrate indicating that this component is not essential for either enzymatic activity or assembly of the enzyme. Mutations in all the other subunits express a respiratory-deficient phenotype and the absence of detectable enzyme activity. Among the respiratory-defective strains, some have mature cytochrome b (non-heme iron protein and cytochrome c1 mutants), while other mutants lack spectrally detectable cytochrome b and have reduced levels of the apoprotein (mutants in the 44-, 40-, 14-, and 11-kDa core subunits). Mutations in single subunits exert different effects on the concentrations of their partner proteins. These may be summarized as follows: 1) No substantial loss in the 44- or 40-kDa core subunits is seen in single mutants; 2) the concentration of cytochrome c1 is also relatively unaffected by mutations in the other subunits except for the cytochrome b mutant which has 60% of the wild type level of cytochrome c1; 3) all the single mutants have only 15-20% of the normal amount of non-heme iron protein; 4) mutations in the non-heme iron protein have no appreciable effect on the concentrations of the other subunits; 5) mutations in single subunits cause parallel decreases in the concentrations of cytochrome b, the 14-, and the 11-kDa subunits. These results indicate that the synthesis or stability of a subset of subunits depends on the presence of other subunit polypeptides of the complex. At present we favor the idea that the observed changes in the concentrations of some subunits are due to higher turnover rates of the proteins in a partially assembled complex. Based on the mutant phenotypes, a tentative model for the assembly of coenzyme QH2-cytochrome c reductase is proposed. According to this model it is envisioned that the subunits interact with one another in the lipid bilayer. Maturation of apocytochrome b occurs after it is assembled with the nonstructural subunits to form a core structure. This intermediate complex interacts with the non-heme iron protein to form the active holoenzyme.
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PMID:Assembly of the mitochondrial membrane system. Analysis of structural mutants of the yeast coenzyme QH2-cytochrome c reductase complex. 284 66

Cardiac mitochondrial NADH dehydrogenase (Cytochrome c reductase, EC1.6.99.3) catalyses the reduction of ferricytochrome c to ferrocytochrome c by NADH. In the presence of the anthracycline anti-tumour drug, adriamycin, electron transfer from NADH is subverted to dioxygen. Using the electron spin resonance technique of spin trapping with the spin trapping agent 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) adriamycin was found to stimulate the formation of superoxide and hydroxyl radicals in the NADH/NADH dehydrogenase reaction. Hydroxyl radical formation is dependent on the availability of trace amounts of redox active metal ions - particularly ferric ions. Trace amounts of ferric ions catalyse the formation of hydroxyl radicals by both superoxide-dependent and adriamycin-dependent one electron reduction of hydrogen peroxide. The metabolism of adriamycin by cardiac mitochondrial NADH dehydrogenase may be an important etiological factor in adriamycin-induced cardiotoxicity. It may be therapeutically beneficial to keep nonessential ferric/ferrous ions in the myocardium at minimum levels with siderophoric iron chelators - providing the anti-tumour activity of adriamycin is not impaired.
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PMID:Reduction of oxygen by NADH/NADH dehydrogenase in the presence of adriamycin. 285 Feb 70

Succinate dehydrogenase is a conserved membrane-bound enzyme consisting of two nonidentical subunits: a flavo iron-sulfur protein (Fp) subunit, containing a covalently bound flavin, and an iron-sulfur protein (Ip) subunit. Bacillus subtilis succinate dehydrogenase in wild type bacteria and 12 well characterized succinate dehydrogenase-defective mutants were examined by low temperature EPR spectroscopy to characterize the enzyme and study subunit location and biosynthesis of its iron-sulfur clusters. The wild type B. subtilis enzyme contains iron-sulfur clusters which are analogous to clusters S-1 and S-3 of bovine heart succinate dehydrogenase but with slightly different EPR characteristics. Spins from cluster S-2 were not detectable as in the case of the intact form of bovine heart succinate dehydrogenase. However, dithionite reduction of the B. subtilis enzyme greatly enhanced spin relaxation of the ferredoxin-type cluster S-1, indicating the presence of the cluster S-2. Iron-sulfur cluster S-1 was found to be assembled in soluble succinate dehydrogenase subunits in the cytoplasm, but only if full-length Fp polypeptides and relatively large fragments of Ip polypeptides were present. Cluster S-1 was not detected in mutants with soluble mutated Fp polypeptides or in a mutant totally lacking Ip subunit polypeptide. Iron-sulfur clusters S-1, S-2, and S-3 were assembled also when the covalently bound flavin in the Fp subunit was absent. Clusters S-1 and S-3 in the membrane-bound flavin-deficient succinate dehydrogenase were not reduced by succinate but could be reduced by electron transfer from NADH dehydrogenase via the menaquinone pool.
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PMID:Characterization by electron paramagnetic resonance and studies on subunit location and assembly of the iron-sulfur clusters of Bacillus subtilis succinate dehydrogenase. 298 99


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