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:1.6.99.1 (NADPH-diaphorase)
3,903 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Cytochemical reactions for mitochondrial NADH-dependent dehydrogenases (diaphorase/NADH which is related to flavoprotein), NAD-dependent dehydrogenases (isocitrate, malate) and succinate dehydrogenase were carried out in rat spermatozoa. In addition to a morphological evaluation, the intensity of the reactions was assessed using a computer image analysing system (Quantimet 600 S). The intensity of the reactions was examined in sperm midpieces by measuring integrated optical density (IOD) and mean optical density (MOD). The activity of mitochondrial respiratory chain complexes was also analysed using the polarographic method. In the population of spermatozoa studied, all whole spermatozoa midpieces were completely filled with formazans, the product of the cytochemical reaction. These morphological findings corresponded to the values obtained for IOD and MOD for the given enzymes. In the oxygraphic studies, the spermatozoa demonstrated consumption of oxygen in the presence of substrates for I, II and IV complexes and their mitochondria revealed normal integrity and sensitivity to the substrates and inhibitors. However, the oxygraphic studies revealed differences between the sperm and somatic cells. These differences concerned the stimulation of pyruvate oxidation by malate, the lack of an effect of malonic acid on phenazine methosulphate (an acceptor of electrons) oxidation and the lack of an effect of cytochrome c on ascorbate oxidation. The cytochemical method, together with densitometric measurements, enables: (1) the reaction intensity to be determined objectively; (2) subtle and dramatic differences in reaction intensity to be revealed between spermatozoa that do not differ under morphological evaluation of the intensity; (3) possible defects within the mitochondrial sheath to be located and assessed in a large number of spermatozoa. This method can be used as a screening method alongside the routine morphological examination of spermatozoa. On the other hand, the oxygraphic method in the inner membrane of mitochondria can reveal functional changes which are related to the action of respiratory chain complexes and display characteristic features of mitochondria energy metabolism. The methods used are complementary and allow the complex evaluation of mitochondria in spermatozoa. Both methods can be used in experimental and clinical studies.
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PMID:Computerized analysis of cytochemical reactions for dehydrogenases and oxygraphic studies as methods to evaluate the function of the mitochondrial sheath in rat spermatozoa. 1116 13

Besides oxidizing L-arginine, neuronal NO synthase (NOS) NADPH-dependently reduces various electron acceptors, including cytochrome c and tetrazolium salts. The latter NADPH diaphorase reaction is used as a NOS-specific histochemical stain. Both reductase activities have been utilized to analyse electron transfer mechanisms within NOS. Basal L-arginine turnover by homodimeric NOS is enhanced by exogenous tetrahydrobiopterin, and the intra-subunit electron flow may include intermediate trihydrobiopterin. In the present work we have investigated the possible role of the tetrahydrobiopterin binding site of NOS in its reductase activities by examining the effects of anti-pterin type (PHS) NOS inhibitors. Although the type I anti-pterin, PHS-32, which does not affect basal dimeric NOS activity, also had no effect on either reductase activity, the type II anti-pterin, PHS-72, which inhibits basal NOS activity, inhibited both reductase activities and the NADPH diaphorase histochemical stain. Pterin-free NOS monomers catalysed both cytochrome c and tetrazolium salt reduction. Our data suggest that both NOS reductase activities are independent of tetrahydrobiopterin. However, occupation of an exosite near the pterin site in NOS by type II anti-pterins may interfere with the electron flow within the active centre, suggesting that steric perturbation of the pterin binding pocket or reductase interaction contribute to the mechanism of inhibition by this class of NOS inhibitors.
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PMID:Pterin interactions with distinct reductase activities of NO synthase. 1133 34

Ferredoxin and ferredoxin-NADP+ oxidoreductase (FNR) were purified from leaves, roots, and red and green pericarp of tomato (Lycopersicon esculentum, cv VFNT and cv Momotaro). Four different ferredoxins were identified on the basis of N-terminal amino acid sequence and charge. Ferredoxins I and II were the most prevalent forms in leaves and green pericarp, and ferredoxin III was the most prevalent in roots. Red pericarp of the VFNT cv yielded variable amounts of ferredoxins II and III plus a unique form, ferredoxin IV. Red pericarp of the Momotaro cv contained ferredoxins I, II, and IV. This represents the first demonstration of ferredoxin in a chromoplast-containing tissue. There were no major differences among the tomato ferredoxins in absorption spectrum or cytochrome c reduction activity. Two forms of FNR were present in tomato as judged by anion exchange chromatography and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. FNR II had a lower apparent relative molecular weight, a slightly altered absorption spectrum, and a lower specific activity for cytochrome c reduction than FNR I. FNR II could be a partially degraded form of FNR I. The FNRs from the different tissues of tomato plants all showed diaphorase activity, with FNR II being more active than FNR I. The presence of ferredoxin and FNR in heterotrophic tissues of tomato is consistent with the existence of a nonphotosynthetic ferredoxin/FNR redox pathway to support the function of ferredoxin-dependent enzymes.
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PMID:Ferredoxin and ferredoxin-NADP reductase from photosynthetic and nonphotosynthetic tissues of tomato. 1153 2

An enzyme exhibiting NADH oxidase (diaphorase) activity was isolated from the hyperthermophilic sulfate-reducing anaerobe Archaeoglobus fulgidus. N-terminal sequence of the protein indicates that it is coded for by open reading frame AF0395 in the A. fulgidus genome. The gene AF0395 was cloned and its product was purified from Escherichia coli. Like the native NADH oxidase (NoxA2), the recombinant NoxA2 (rNoxA2) has an apparent molecular mass of 47 kDa, requires flavin adenine dinucleotide for activity, has NADH-specific activity, and is thermostable. Hydrogen peroxide is the product of bivalent oxygen reduction by rNoxA2 with NADH. The rNoxA2 is an oxidase with diaphorase activity in the presence of electron acceptors such as tetrazolium and cytochrome c. During purification NoxA2 remains associated with the enzyme responsible for D-lactate oxidation, the D-lactate dehydrogenase (Dld), and the genes encoding NoxA2 and Dld are in the same transcription unit. Together these results suggest that NADH oxidase may be involved in electron transfer reactions resulting in sulfate respiration.
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PMID:H(2)O(2)-forming NADH oxidase with diaphorase (cytochrome) activity from Archaeoglobus fulgidus. 1171 57

Previously, we hypothesized that hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) can be biotransformed by anaerobic sludge via three different routes: (1) direct ring cleavage via alpha-hydroxylation of a-CH(2) group, (2) reduction of one of the -NO(2) groups to -NO, (3) N-denitration prior to ring cleavage. The present study describes biotransformation of RDX via route 3 by a diaphorase (EC 1.8.1.4) from Clostridium kluyveri using NADH as electron donor. The removal of RDX was accompanied by the formation and accumulation of nitrite ion (NO(2)(-)), formaldehyde (HCHO), ammonium (NH(4)(+)), and nitrous oxide (N(2)O). None of the RDX-nitroso products were detected. The ring cleavage product methylenedinitramine was detected as the transient intermediate. Product stoichiometry showed that each reacted RDX molecule produced one nitrite ion and the product distribution gave a carbon (C) and nitrogen (N) mass balance of 91 and 92%, respectively, supporting the occurrence of a mono-denitration step prior to the ring cleavage and decomposition. Severe oxygen mediated inhibition (92% inhibition) of RDX biotransformation and superoxide dismutase-sensitive cytochrome c reduction indicated the potential involvement of an anion radical RDX(.-) prior to denitration. A comparative study between native- and apo-enzymes showed the possible involvement of flavin mononucleotide (FMN) in catalyzing the transfer of a redox equivalent (e/H(+)) from NADH to RDX to produce RDX(.-) responsible for secondary decomposition.
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PMID:Diaphorase catalyzed biotransformation of RDX via N-denitration mechanism. 1220 Jan 15

Downey, Ronald J. (University of Notre Dame, Notre Dame, Ind.). Vitamin K-mediated electron transfer in Bacillus subtilis. J. Bacteriol. 88:904-911. 1964.-Electron transfer enzymes were obtained from log-phase cells of Bacillus subtilis after aerobic and anaerobic cultivation. The cytochrome content was found to be related to oxygen tension, there being little, if any, cytochrome operative in anaerobic cells. Vitamin K levels in the two cell types did not vary as markedly. A soluble diaphorase-type flavoprotein was obtained from both types of cells which reacted with vitamin K(2), K(3), and certain dyes but not bovine cytochrome c. Almost 90% of this diaphorase activity was leached from intact protoplasts without the use of solvating agents or sonic oscillation. Electron transport particles capable of coupled phosphorylation were inhibited by light (360 mmu) or 2,3-dimercaptopropanol (BAL), whereas these had no effect on the diaphorase activity. Phosphorylation in a BAL-inhibited system was restored after addition of the soluble diaphorase from either aerobic or anaerobic cells. The results suggested that soluble flavoprotein components are linked to vitamin K in both fermentative and phosphorylative pathways, and that this segment is indispensable to aerobic and anaerobic respiration in the bacillus.
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PMID:VITAMIN K-MEDIATED ELECTRON TRANSFER IN BACILLUS SUBTILIS. 1421 53

To study the role of endogenous NO in survival and recovery of spinal cord neurons after nerve lesions, wild type mice were compared to knock-out mice lacking neuronal, endothelial or inducible NO synthase (NOS) after sciatic nerve transection. The NO-generating capacities were assessed by NOS immunohistochemistry and NADPH-diaphorase staining. The feature of affected neurons was evaluated following Nissl- and TUNEL-staining, by immunocytochemical demonstration of cytochrome c-translocation, and by ultrastructural examination. Time point of cell loss was found to be independent of the mice type and occurred only at later post-axotomy states. The extent of neuronal degeneration, however, depended on the NO supply. Whereas a lack of endothelial or inducible NOS was well tolerated, deficiency of neuronal NOS enhanced the competence-to-die and led to a substantial apoptotic cell death of spinal cord neurons. Thus, NO supply turned out to be essential for cell survival and recovery with reference to the neuronal NOS isoform.
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PMID:Neuronal NOS deficiency promotes apoptotic cell death of spinal cord neurons after peripheral nerve transection. 1513 63

From Bacillus subtilis cell extracts, ferredoxin-NADP+ reductase (FNR) was purified to homogeneity and found to be the yumC gene product by N-terminal amino acid sequencing. YumC is a approximately 94-kDa homodimeric protein with one molecule of non-covalently bound FAD per subunit. In a diaphorase assay with 2,6-dichlorophenol-indophenol as electron acceptor, the affinity for NADPH was much higher than that for NADH, with Km values of 0.57 microM vs >200 microM. Kcat values of YumC with NADPH were 22.7 s(-1) and 35.4 s(-1) in diaphorase and in a ferredoxin-dependent NADPH-cytochrome c reduction assay, respectively. The cell extracts contained another diaphorase-active enzyme, the yfkO gene product, but its affinity for ferredoxin was very low. The deduced YumC amino acid sequence has high identity to that of the recently identified Chlorobium tepidum FNR. A genomic database search indicated that there are more than 20 genes encoding proteins that share a high level of amino acid sequence identity with YumC and which have been annotated variously as NADH oxidase, thioredoxin reductase, thioredoxin reductase-like protein, etc. These genes are found notably in gram-positive bacteria, except Clostridia, and less frequently in archaea and proteobacteria. We propose that YumC and C. tepidum FNR constitute a new group of FNR that should be added to the already established plant-type, bacteria-type, and mitochondria-type FNR groups.
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PMID:Purification and characterization of ferredoxin-NADP+ reductase encoded by Bacillus subtilis yumC. 1525 6

The photochemical activities of chloroplasts isolated from bundle sheath and mesophyll cells of maize (Zea mays var. DS606A) have been measured. Bundle sheath chloroplasts are almost devoid of grana, except in very young leaves, while mesophyll chloroplasts contain grana at all stages of leaf development.Chloroplast fragments isolated from bundle sheath cells showed a light-dependent reduction of potassium ferricyanide, 2, 6-dichlorophenolindophenol, mammalian cytochrome c, plastocyanin, and Euglena cytochrome c(552). These activities were inhibited by 3-(3, 4-dichlorophenyl)-1, 1-dimethylurea at 1.25 micromolar. However, the photoreduction of NADP from water was extremely low or absent, except in chloroplasts from very young leaves, and the capacity for NADP reduction appeared to be related to the degree of grana formation.Photosystem I activity was present in bundle sheath chloroplast preparations at all stages of leaf growth and senescence examined. However, the activity was lower than in isolated mesophyll chloroplasts. NADPH diaphorase activity was comparable in both types of chloroplast.Chloroplasts isolated from bundle sheath cells of plants grown under a variety of conditions, including continuous and intermittent light, high and low light intensities, and high temperature, exhibited photosystem II activity.
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PMID:Photosystem II Activity in Agranal Bundle Sheath Chloroplasts from Zea mays. 1665 84

THE ASSIMILATORY NITRATE REDUCTASE (NADH: nitrate oxidoreductase, E.C. 1.6.6.2.) from the marine diatom Thalassiosira pseudonana, Hasle and Heimdal, has been purified 200-fold and characterized. The regulation of nitrate reductase in response to various conditions of nitrogen nutrition has been investigated.Nitrate reductase activity is repressed by the presence of ammonium in vivo, and its synthesis is derepressed when ammonium is absent. The derepression process is sensitive to cycloheximide and apparently requires protein synthesis. Repression of enzyme activity by ammonium is neither inhibited nor delayed by the presence of cycloheximide. In vitro, ammonium does not inhibit enzyme activity.NADH is the physiological electron donor for the enzyme in a flavin-dependent reaction. Spectral studies have indicated the presence of a b-type cytochrome associated with the enzyme. It is possible to observe enzymatic oxidation-reduction reactions which represent partial functions of the over-all electron transport capacity of this enzyme. Nitrate reductase will accept electrons from artificial electron donors such as reduced methyl viologen in a flavin-independent reaction. Further, dithionitereduced flavin adenine dinucleotide can donate electrons to the enzyme to reduce nitrate to nitrite. Finally, the nitrate reductase will exhibit a diaphorase activity and reduce the artificial electron acceptor mammalian cytochrome c in flavin-adeninedinucleotide-dependent reaction.Inhibition studies with potassium cyanide, sodium azide, and o-phenanthroline have yielded indirect evidence for metal component (s) of the enzyme.The inhibition of the NADH-requiring enzyme activities by p-hydroxymercuribenzoate has shown that an essential sulfhydryl group is involved in the initial portion of the electron transport. Heat treatment exerts an effect similar to the p-hydroxymercuribenzoate inhibition; namely, the NADH-requiring activities are rapidly inactivated, whereas the terminal nitrate-reducing activities are relatively stable to heat.The T. pseudonana nitrate reductase molecule has the hydrodynamic properties of an ellipsoid with a frictional coefficient of 1.69 and a molecular weight of 330,000.
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PMID:Purification and Characterization of the Nitrate Reductase from the Diatom Thalassiosira pseudonana. 1665 41


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