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)

To investigate the functional role of the cysteine residues present in the spinach ferredoxin-NADP+ oxidoreductase, we individually replaced each of the five cysteine residues with serine using site-directed mutagenesis. All of the mutant reductases were correctly assembled in Escherichia coli except for the C42S mutant protein. C114S and C137S mutant enzymes apparently showed structural and kinetic properties very similar to those of the wild-type reductase. However, C272S and C132S mutations yielded enzymes with a decreased catalytic activity in the ferredoxin-dependent reaction (14 and 31% of the wild type, respectively). Whereas the C132S was fully competent in the diaphorase reaction, the C272S mutant flavoprotein showed a 35-fold reduction in catalytic efficiency with respect to the wild-type enzyme (0.4 versus 14.28 microM-1 s-1) due to a substantial decrease of kcat. NADP+ binding by the C272S mutant enzyme was apparently quantitatively the same (Kd = 37 microM) but qualitatively different, as shown by the differential spectrum. Stopped-flow experiments showed that the enzyme-FAD reduction rate was considerably decreased in the C272S mutant reductase, along with a much lower yield of the charge-transfer transient species. It is inferred from these data that the charge transfer (FAD-NADPH) between the reductase and NADPH is required for hydride transfer from the pyridine nucleotide to flavin to occur with a rate compatible with catalysis.
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PMID:The role of cysteine residues of spinach ferredoxin-NADP+ reductase As assessed by site-directed mutagenesis. 851 83

Incubation of either Chlorella nitrate reductase or the recombinant flavin domain of spinach nitrate reductase with reagents specific for modification of cysteine residues, such as N-ethylmaleimide, resulted in a time-dependent inactivation of NADH:ferricyanide reductase activity which could be prevented by incubation in the presence of NADH. At 25 degrees C and employing a fixed enzyme:modifier ratio, the rate of inactivation for both the Chlorella and spinach enzymes followed the order p-chloromercuribenzoate > methyl methanethiosulfonate > 2-(4'-maleimidylanilino)naphthalene-6-sulfonic acid > N-ethylmaleimide. For the spinach flavin domain, inactivation by methyl methanethiosulfonate or p-chloromercuribenzoate was found to be concentration independent suggesting the absence of nonspecific modifications. Initial rate studies of the methyl methanethiosulfonate-modified flavin domain indicated a reduction in NADH:ferricyanide activity (Vmax) from 85 to 44 micromol NADH consumed/min/nmol FAD and an increase in the Km for NADH from 12 to 35 microM when compared to the native enzyme, confirming a role for cysteine residue(s) in maintaining diaphorase activity. Site-directed mutagenesis of the four individual cysteines (residues 17, 54, 62, and 240) in the recombinant spinach flavin domain resulted in mutant proteins with visible and CD spectra very similar to those of the wild-type domain. Initial rate studies indicated that only substitutions of serine for cysteine 240 decreased diaphorase activity with maximal NADH:ferricyanide activity for the C240S mutant corresponding to 51 micromol NADH consumed/min/nmol FAD with a Km for NADH of 14 microM. Mutation of C240 to Ala or Gly resulted in greater loss of activity. The thermal stability of the four serine mutants was slightly decreased compared to the wild-type domain with the C62S mutant exhibiting the greatest instability. In contrast to the effects on diaphorase activity, square wave voltammetric studies indicated changes in the oxidation-reduction midpoint potential for the FAD/FADH2 couple in the C54S (E0'= -197 mV), C62S (E0' = -226 mV), and C240S (E0' = -219 mV) mutants compared to the wild-type domain (E0' = -268 mV). These results indicate that of the four cysteine residues in the spinach nitrate reductase flavin domain, only C240 plays a role in maintaining diaphorase activity, while C54 has the greatest influence on flavin redox potential and that no correlation between changes in catalytic activity and flavin redox potential was observed.
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PMID:Thiol modification and site directed mutagenesis of the flavin domain of spinach NADH:nitrate reductase. 866 Jun 90

The inhibition of mitochondrial succinate cytochrome c reductase by nordihydroguaiaretic acid (NDGA) was measured in vitro under aerobic and hypoxic conditions. The photodynamic effect of NDGA on EMT6 mouse mammary carcinoma cells was also investigated in vitro under aerobic and hypoxic conditions. NDGA strongly inhibited succinate cytochrome c reductase, which contains the mitochondrial electron transport Complexes II and III. By comparing the I50 values of aerobic and hypoxic assays, NDGA inhibition was found to be oxygen-independent. This finding suggests that oxygen is not involved in the inhibitory effect of mitochondrial respiration by NDGA. Similarly, our data demonstrate that NDGA-induced cytotoxicity towards EMT6 tumor cells is neither light-dependent nor oxygen-dependent. A dose-dependent loss of viability of EMT6 cell was observed although the toxicity of NDGA towards EMT6 cells was not strong, with an LC50 in the 200 microM range. NDGA caused a depletion of mitochondrial sulfhydryl groups. Sulfhydryl compounds, GSH and cysteine, prevented the inhibition of succinoxidase activity by NDGA. This suggests that NDGA exerts its biological effects by the depletion of sulfhydryl groups in key biochemical systems related to mitochondrial function.
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PMID:Effect of NDGA on beef heart mitochondria and EMT6 mouse mammary carcinoma cells. 874 92

We examined whether highly conserved cysteine residues in the reductase domain of the constitutive isoform of nitric oxide synthase in human endothelial cells (ecNOS) are crucial for catalytic activity of the enzyme. Substitution of alanine for cysteines 976 (Cys-976), 991 (Cys-991), 1048 (Cys-1048), or 1114 (Cys-1114), located in the reductase domain of human ecNOS, was achieved by oligonucleotide-directed mutagenesis and expression in COS-7 cells. The specific activity of ecNOS was > 7-fold increased in wild-type and in mutants Cys-976 and Cys-991, but not in mutants Cys-1048 and Cys-1114. However, Western blot analysis indicated that expression of ecNOS protein was comparable in wild-type and in all mutants. NADPH concentration-dependent L-citrulline formation and NADPH oxidation during L-arginine metabolism were reduced in mutants Cys-1048 and Cys-1114 compared to wild-type. Similarly, NADPH cytochrome c reductase activity was increased in a time-dependent fashion in wild-type but not in mutants Cys-1048 and Cys-1114. These results indicate that Cys-1048 and Cys-1114 residues in the NADPH binding site of the reductase domain are critical for human ecNOS activity. The lack of utilization of NADPH in L-arginine metabolism and in cytochrome c reduction suggests that these active site cysteine residues may be responsible for binding of NADPH and/or for electron transfer in human ecNOS.
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PMID:Reductase domain cysteines 1048 and 1114 are critical for catalytic activity of human endothelial cell nitric oxide synthase as probed by site-directed mutagenesis. 880 29

NADH dehydrogenase in the plasma membrane transfers electrons from NADH to external oxidants like ferricyanide, through pathways which are linked to metabolic processes in the cell. Hormone binding to specific sites (receptors) can modify the enzyme activity, suggesting a direct or indirect coupling between the redox system and the hormone receptors. Reduction of external ferricyanide to ferrocyanide by human erythrocytes was stimulated by beta-adrenergic agonists (adrenaline, ritodrine and isoxsuprine), this effect being dependent upon concentration and pH. The agonist-stimulatory effect was attenuated in the presence of metoprolol (10(-4) M), a beta-adrenergic antagonist, and was not modified in the presence of prazosin, an alpha-adrenergic antagonist, suggesting that modification of the redox activity is mediated by binding of the agonists to beta-adrenergic receptors present in the human erythrocytes. Basal and agonist-dependent activities were inhibited in the presence of sulfhydryl reagents p-chloromercuribenzoate (PCMB, 10(-5) M) and N-ethylmaleimide (NEM, 10(-3) M), indicating the involvement of -SH groups. Inactivation by NEM was reversed by washing the cells with GTP (10(-3) M) and GTP gamma S (10(-4) M), suggesting that the specific alkylated -SH group(s) is located on a G protein in the hormone-receptor-G-protein complex. The human erythrocytes contain G proteins, displaying both guanine-nucleotide-binding properties and GTPase activity. Fluoride (10(-2) M) and fluoroaluminate (AlF4- (F-, 10(-2) M + Al3+, 10(-5) M), G protein activators, enhanced the basal and agonist-dependent activities, suggesting the involvement of G proteins in this system. The overall results indicated that one of the coupling components between the hormonal receptors and the redox system is probably a G protein, and the mechanism of enzyme activation after hormone binding to the receptor is based on the redox state of cysteine residues probably within the receptor-G-protein complex.
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PMID:Activation of a NADH dehydrogenase in the human erythrocyte by beta-adrenergic agonists: possible involvement of a G protein in enzyme activation. 926 92

Mitochondrial mRNAs in trypanosomatids are edited by uridylate insertion and deletion. The respiratory chain complexes cytochrome c reductase, cytochrome c oxidase and F0F1-ATPase of the insect trypanosomatid Crithidia fasciculata have been isolated and analysed by peptide microsequencing, but so far, proteins encoded by edited (and unedited) mitochondrial mRNAs have not been found. In this paper, we provide evidence that the mitochondrial mRNAs encoding the three large subunits of cytochrome c oxidase are indeed translated. First, purified holo cytochrome c oxidase turned out to be cysteine-rich, in agreement with the high cysteine codon-content of the sequence of mitochondrial cox subunit mRNAs. Second, in mass spectrometry measurements of cytochrome c oxidase, a protein was detected with the predicted molecular weight of cytochrome c oxidase subunit 2. Finally, an antibody generated against a fusion protein produced in Escherichia coli from constructs containing a segment of cytochrome c oxidase subunit 2 cDNA, specifically recognised protein bands present in cytochrome c oxidase following SDS PAGE. However, these proteins were present in the high molecular weight region of the gel, suggesting that cytochrome c oxidase subunit 2 aggregates in the presence of SDS.
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PMID:Further evidence for the presence of mitochondrially encoded subunits in cytochrome c oxidase of the trypanosomatid Crithidia fasciculata. 937 79

Oxygen radical generating systems, namely, Cu(II)/ H2O2, Cu(II)/ascorbate, Cu(II)/NAD(P)H, Cu(II)/ H2O2/catecholamine and Cu(II)/H2O2/SH-compounds irreversibly inhibited yeast glutathione reductase (GR) but Cu(II)/H2O2 enhanced the enzyme diaphorase activity. The time course of GR inactivation by Cu(II)/H2O2 dependent on Cu(II) and H2O2 concentrations and was relatively slow, as compared with the effect of Cu(II)/ascorbate. The fluorescence of the enzyme Tyr and Trp residues was modified as a result of oxidative damage. Copper chelators, catalase, bovine serum albumin and HO. scavengers prevented GR inactivation by Cu(II)/H2O2 and related systems. Cysteine, N-acetylcysteine, N-(2-dimercaptopropionylglycine and penicillamine enhanced the effect of Cu(II)/H2O2 in a concentration- and time-dependent manner. GSH, Captopril, dihydrolipoic acid and dithiotreitol also enhanced the Cu(II)/H2O2 effect, their actions involving the simultaneous operation of pro-oxidant and antioxidant reactions. GSSG and trypanothione disulfide effectively protected GR against Cu(II)/H2O2 inactivation. Thiol compounds prevented GR inactivation by the radical cation ABTS.+. GR inactivation by the systems assayed correlated with their capability for HO. radical generation. The role of amino acid residues at GR active site as targets for oxygen radicals is discussed.
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PMID:Inactivation of yeast glutathione reductase by Fenton systems: effect of metal chelators, catecholamines and thiol compounds. 945 90

Previous studies from our laboratory have shown that mitochondrial dysfunction may be an important early event in S-[(1 and 2)-phenyl-2-hydroxyethyl]cysteine (PHEC)-induced cytotoxicity in isolated rat renal proximal tubules. The present study has therefore examined in more detail PHEC-induced mitochondrial dysfunction, both in vivo and in vitro, using isolated renal cortical mitochondria. Renal cortical mitochondria isolated from PHEC-treated rats in vivo showed depressed effects on the mitochondrial respiration and oxidative phosphorylation in both a dose (0, 250, and 500 micromol/kg iv)- and time (0-24 h)-dependent manner in the presence of both succinate (Site 2) and malate plus alpha-ketoglutarate (Site 1) as respiratory substrates, with initial significant depression occurring as early as 4 h following treatment with 500 micromol PHEC/kg. Similar mitochondrial dysfunctions were observed in vitro in concentration- and time-dependent manners with both respiratory substrates. PHEC also caused a marked dose-dependent inhibition of mitochondrial succinate dehydrogenase and NADH cytochrome c reductase activities both in vivo and in vitro, with initial inhibition occurring as early as 4 h after in vivo administration and 45 min after exposure to PHEC in vitro, while the NADH dehydrogenase activity was not considerably inhibited. The mitochondrial ATPase activity was significantly decreased 4 and 24 h following treatment with PHEC (500 micromol/kg). These results suggest that PHEC exerts its inhibitory effect on the mitochondrial respiration and oxidative phosphorylation through the action on the mitochondrial electron transport chain. PHEC significantly reduced the activity of adenine nucleotide translocase as well as the net uptake of substrates by mitochondria without affecting their efflux within 2-4 h after its injection (500 micromol/kg). On the other hand, significant renal damage, as assessed by morphological study, appeared as early as 24 h following such treatment. The observation of similar effects after both in vivo and in vitro exposures may suggest that the effect on mitochondria may have a pathogenic role in PHEC-induced renal injury in rats. PHEC produces mitochondrial toxicity that results from an inactivation of mitochondrial anionic substrate transporters as well as from an inhibition of activities of adenine nucleotide translocase and dehydrogenases.
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PMID:S-[(1 and 2)-phenyl-2-hydroxyethyl]cysteine-induced alterations in renal mitochondrial function in male Fischer-344 rats. 970 95

1. The distribution and localization of nitric oxide synthase (NOS) immunoreactivity and nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) activity in the bovine oesophageal groove were investigated by immnunohistochemical and histochemical staining techniques. Functional in vitro studies were performed to correlate the presence of NOS-immunoreactivity (IR) and NADPH-d staining with smooth muscle relaxations involving the L-arginine/nitric oxide neural pathway in the bovine oesophageal groove activity. 2. NOS-IR and NADPH-d were expressed in nerve cell bodies of the myenteric, submucosal and intramuscular ganglia, and in nerve fibres distributed around blood vessels and throughout the different muscular layers of the bovine oesophageal groove. 3. In oesophageal groove strips treated with guanethidine (10(-5) M) and atropine (10(-7) M) to block noradrenergic neurotransmission and muscarinic receptors, respectively, electrical field stimulation (EFS, 0.5-32 Hz, 1 ms duration, 20-s trains) induced relaxations which were practically abolished by tetrodotoxin (TTX, 10(-6) M). 4. Incubation with an inhibitor of nitric oxide synthesis, NG-nitro-L-arginine (L-NOARG, 3 x 10(-5) M), significantly inhibited relaxations induced by EFS. This inhibition was partially reversed by L-arginine (L-arg, 5 x 10(-3) M). D-NOARG (3 x 10(-5) M) had no effect on EFS-induced relaxations. 5. NO added as an acidified solution of NaNO2 (10(-6) - 10(-3) M) and S-nitroso-L-cysteine (10(-7) - 10(-4) M) caused concentration-dependent relaxations of the bovine oesophageal groove. These relaxations were unaffected by L-NOARG (3 x 10(-5) M). 6. The presence of NO-synthesizing enzyme in nerves and ganglia, and the pharmacological evidence for NO-mediated smooth muscle relaxation suggested that the L-arg/NO neuronal pathway is involved in the inhibitory neurotransmission of the bovine oesophageal groove.
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PMID:Involvement of the L-arginine/nitric oxide neural pathway in non-adrenergic, non-cholinergic relaxation of the bovine oesophageal groove. 973 Feb 60

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


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