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)

1. In Aspergillus nidulans nitrate and nitrite induce nitrate reductase, nitrite reductase and hydroxylamine reductase, and ammonium represses the three enzymes. 2. Nitrate reductase can donate electrons to a wide variety of acceptors in addition to nitrate. These artificial acceptors include benzyl viologen, 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyltetrazolium chloride, cytochrome c and potassium ferricyanide. Similarly nitrite reductase and hydroxylamine reductase (which are possibly a single enzyme in A. nidulans) can donate electrons to these same artificial acceptors in addition to the substrates nitrite and hydroxylamine. 3. Nitrate reductase can accept electrons from reduced benzyl viologen in place of the natural donor NADPH. The NADPH-nitrate-reductase activity is about twice that of reduced benzyl viologen-nitrate reductase under comparable conditions. 4. Mutants at six gene loci are known that cannot utilize nitrate and lack nitrate-reductase activity. Most mutants in these loci are constitutive for nitrite reductase, hydroxylamine reductase and all the nitrate-induced NADPH-diaphorase activities. It is argued that mutants that lack nitrate-reductase activity are constitutive for the enzymes of the nitrate-reduction pathway because the functional nitrate-reductase molecule is a component of the regulatory system of the pathway. 5. Mutants are known at two gene loci, niiA and niiB, that cannot utilize nitrite and lack nitrite-reductase and hydroxylamine-reductase activities. 6. Mutants at the niiA locus possess inducible nitrate reductase and lack nitrite-reductase and hydroxylamine-reductase activities. It is suggested that a single enzyme protein is responsible for the reduction of nitrite to ammonium in A. nidulans and that the niiA locus is the structural gene for this enzyme. 7. Mutants at the niiB locus lack nitrate-reductase, nitrite-reductase and hydroxylamine-reductase activities. It is argued that the niiB gene is a regulator gene whose product is necessary for the induction of the nitrate-utilization pathway. The niiB mutants either lack or produce an incorrect product and consequently cannot be induced. 8. Mutants at the niiribo locus cannot utilize nitrate or nitrite unless provided with a flavine supplement. When grown in the absence of a flavine supplement the activities of some of the nitrate-induced enzymes are subnormal. 9. The growth and enzyme characteristics of a total of 123 mutants involving nine different genes indicate that nitrate is reduced to ammonium. Only two possible structural genes for enzymes concerned with nitrate utilization are known. This suggests that only two enzymes, one for the reduction of nitrate to nitrite, the other for the reduction of nitrite to ammonium, are involved in this pathway.
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PMID:Genetic and biochemical studies of nitrate reduction in Aspergillus nidulans. 438 27

Crude extracts of Methanospirillum hungatei strain GP1 contained NADH and NADPH diaphorase activities. After a 483-fold purification of the NADH diaphorase the enzyme was further separated from contaminating proteins by polyacrylamide disc gel electrophoresis. Two distinct activity bands were extracted from the acrylamide, each one having oxygen, 2,6-dichlorophenolindophenol, and cytochrome c linked activities. In these preparations NADPH could not replace NADH as electron donor. During the initial purification steps all activity was lost due to the removal of a readily released cofactor. Enzyme activity was restored by either FAD or a FAD fraction isolated from M. hungatei. Oxidase activity exhibited a broad pH optimum from 7.0 to 8.5 and apparent Km values of 26 microM for NADH and 0.2 microM for FAD. Superoxide anion, formed in the presence of oxygen, accounted for all of the NADH consumed in the reaction. The molecular weight of the diaphorase was about 117 500 by sodium dodecyl sulfate gel electrophoresis. Sulfhydryl reagents and chelating agents were inhibitory. Inactivation, which occurred during storage in phosphate buffer at 4 degrees C, was delayed by dithiothreitol. The isolated NADH diaphorase lacked NADPH:NAD transhydrogenase and NAD reductase activities.
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PMID:Isolation and characterization of a FAD-dependent NADH diaphorase from Methanospirillum hungatei strain GP1. 626 28

The kinetic characteristics of the diaphorase activities associated with the NADH-dependent nitrite reductase (EC 1.6.6.4) from Escherichia coli have been determined. The values of the apparent maximum velocity are similar for the reduction of Fe(CN)6(3)-and mammalian cytochrome c by NADH. These reactions may therefore have the same rate-limiting step. NAD+ activates NADH-dependent reduction of cytochrome c, and the apparent maximum velocity for this substrate increases more sharply with the concentration of NAD+ than for hydroxylamine. The simplest explanation is that NAD+ activation of hydroxylamine reduction derives solely from activation of steps involved in the reduction of cytochrome c, a flavin-mediated reaction, but these steps are only partly rate-limiting for the reduction of hydroxylamine. At 0.5 mM-NAD+, the apparent maximum velocity was 2.3 times higher for 0.1 mM-cytochrome c as substrate than for 100 mM-hydroxylamine, suggesting that the rate-limiting step during hydroxylamine reduction is a step that is not involved in cytochrome c reduction. A scheme is proposed that can account for the pattern of variation with [NAD+] of the Michaelis-Menten parameters for hydroxylamine and for NADH with hydroxylamine or cytochrome c as oxidized substrate.
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PMID:The steady state kinetics of the NADH-dependent nitrite reductase from Escherichia coli K12. The reduction of single-electron acceptors. 628 3

The mechanism(s) of natural killer (NK) cell-mediated cytotoxicity (CMC) remains largely unknown. In this study, we investigated the possibility of human NK cells to exhibit an oxidative burst (OB) after stimulation by K562, an NK-sensitive target cell (TC). The addition of catalase (CAT) or superoxide dismutase (SOD) to the NK-mediated cytotoxic assay had no effect on NK-CMC. In contrast, CAT and SOD effectively modulated the cytotoxicity mediated by phorbol-12-myristate-13-acetate (PMA)-activated polymorphonuclear leukocytes (PMNL) against three different tumor TC, including K562. CAT abrogated, while SOD enhanced PMA-activated PMNL-mediated cytotoxicity. The synergistic effect of SOD and PMA was suppressed in a dose-dependent fashion by CAT. Furthermore, by chemiluminescence (CL) and SOD-inhibitable reduction of cytochrome c, we failed to detect an OB associated with K562-stimulated NK cells. PMNL, however, rapidly responded to PMA (10 ng/ml), generating almost 10(6) cpm within 20 min and 26.7 nM O-2/10(6) cells/30 min, as detected by CL and reduction of cytochrome c, respectively. Finally, K562 alone, at cell concentrations corresponding to effector cell:target cell (EC:TC) ratios of 1:1 and 1:10, reduced cytochrome c, but this reduction was not inhibited by SOD, thus suggesting a diaphorase activity. Overall, we show that: a) tumor cell destruction by human NK cells and by PMA-activated PMNL is mediated by different mechanisms; and b) NK-CMC against a sensitive TC does not involve an OB.
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PMID:Compared mechanisms of tumor cytolysis by human natural killer cells and activated polymorphonuclear leukocytes. 632 20

The water-soluble carbodiimide, N-ethyl-3-(3-dimethylaminopropyl)carbodiimide was found to effectively cross-link ferredoxin to ferredoxin-NADP+ reductase. The covalent complex has a stoichiometry of 1 mol of ferredoxin per mol of the reductase. The flavoprotein moiety of the cross-linked complex maintains most of its diaphorase activity and more interestingly has gained the capacity to catalyze the NADPH-cytochrome c reaction without addition of free ferredoxin in the assay mixture. Furthermore, the cross-linked complex binds NADP+ with a Kd = 88 microM at an ionic strength of 0.02 M. These results show that a ternary complex among the reductase and its substrates can be formed, suggesting that the binding sites for ferredoxin and the pyridine nucleotides are distinct. The bound ferredoxin can interact with cytochrome c; the iron-sulfur cluster of the cross-linked complex is shown to be reduced under anaerobic conditions by NADPH and to be required for the catalysis of the NADPH-cytochrome c reductase reaction. The cross-linked complex, added to thylakoids inhibited by the antibody against the reductase, catalyzes the H2O-cytochrome c photoreduction, which suggests that the ferredoxin moiety of the complex can interact with its electron donor in the photosynthetic chain. Restoration of NADP+ photoreduction requires the addition of free ferredoxin.
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PMID:A cross-linked complex between ferredoxin and ferredoxin-NADP+ reductase. 672 48

Spinach ferredoxin was trinitrophenylated by reaction with 2,4,6-trinitrobenzenesulfonate. Four amino groups in the ferredoxin could be modified of the total of five amino groups. The trinitrophenylated ferredoxin formed a complex with ferredoxin-NADP+ reductase just as native ferredoxin did. The modified ferredoxin also retained the activity of electron transport in the cytochrome c photoreduction system of chloroplasts, but could neither donate electrons to ferredoxin-NADP+ reductase in the NADP+ photoreduction system, nor accept electrons from the reductase in the NADPH-cytochrome c reduction system in vitro. Furthermore, it lost the inhibitory effect against the NADPH-diaphorase activity of the reductase. These results suggest that the complex formation of ferredoxin with ferredoxin-NADP+ reductase is a phenomenon essentially independent of the function of electron transport between the two proteins.
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PMID:Trinitrophenylation of spinach ferredoxin and its effect on the functions. 688 43

D-Lactate dehydrogenase, the starting enzyme for carbon and energy metabolism in dissimilatory sulfate-reducing bacteria, has been purified 36-fold from the soluble fraction of the sonicate of Desulfovibrio vulgaris, Miyazaki. The enzyme is specific for D-lactate (Km = 0.8 mM) and DL-2-hydroxybutyrate (probably its D-isomer) as the electron donor substrate. It reduces, in the presence of lactate, various artificial electron acceptors such as 1-methoxyphenazinium methyl sulfate, ferricyanide, tetrazolium dyes, methylene blue, and 2,6-dichlorophenol-indophenol. When 2 mol of ferricyanide was reduced, 1 mol of pyruvate was produced during the reaction. Among natural electron carriers, only cytochrome c-553 isolated from the same organism can be reduced by the enzyme. The ferric complex of pyridine-2,6-dicarboxylate can act as an electron acceptor if cytochrome c-553 is present in the reaction system. NAD+, NADP+, FAD, FMN, cytochrome c3, high-molecular-weight cytochrome, eucaryotic cytochromes c (yeast and horse) and O2 could not be reduced. The enzyme does not have any diaphorase activity. The D-lactate dehydrogenase of D. vulgaris must therefore be named D-lactate:ferricytochrome c-553 oxidoreductase [EC subclass 1.1.2]. A similar enzyme exists in the formate dehydrogenase-less mutant of D. vulgaris, Miyazaki, and in D. vulgaris, Hildenborough.
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PMID:D-lactate dehydrogenase of Desulfovibrio vulgaris. 727 46

NADPH diaphorase activity was found in membrane of DMSO-induced differentiated human promyelocytic leukemia HL-60 cells. This membrane-bound diaphorase activity increased dramatically during differentiation of HL-60 cells. A dye reductase was extracted from membrane of DMSO-induced differentiated HL-60 cells with n-octyl glucoside and sodium cholate in the presence of several protease inhibitors such as PMSF, DIFP, TLCK, antipain, chymostatin, leupeptin, pepstatin A and trypsin inhibitor. The NADPH diaphorase was highly purified by two-stage sequential column chromatographies. The purified enzyme, showing both SOD-insensitive cytochrome c and NBT reductase activities, migrated with an apparent molecular mass of 77 kDa on SDS-PAGE. When the purification of this diaphorase was carried out in the presence of only three protease inhibitors, PMSF, DIFP and TLCK, a partially proteolyzed form of the diaphorase with a molecular mass of 68 kDa was prepared. The proteolyzed diaphorase exhibited only an NADPH-dependent cytochrome c reductase. The NADPH diaphorase gave a positive cross-reaction to polyclonal antibodies raised against microsomal NADPH-cytochrome P450 reductase from rabbit liver.
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PMID:Purification of an NADPH-dependent diaphorase from membrane of DMSO-induced differentiated human promyelocytic leukemia HL-60 cells. 769 24

Soluble extracts of Escherichia coli contain four NADPH:paraquat diaphorases that were separable by anion-exchange HPLC over Mono Q. One of these was induced when the cells were exposed to paraquat. This was the case in a soxRS-competent strain but not in a soxRS-null strain, while a soxRS-constitutive strain overexpressed this diaphorase without the stimulus of exposure to paraquat. This NADPH:paraquat diaphorase could use cytochrome c or nitroblue tetrazolium as an electron acceptor, whereas O2 was a relatively poor acceptor. This diaphorase was identified as the NADPH:ferredoxin reductase. A role for reduced ferredoxin and flavodoxin in the adaptive soxRS response to oxidative stress and in the regulation of the redox status of soxR is discussed.
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PMID:NADPH: ferredoxin oxidoreductase acts as a paraquat diaphorase and is a member of the soxRS regulon. 810 11

Molecular oxygen or cytochrome c has been described as the electron acceptor of the reaction of old yellow enzyme with NADPH. In this study, menadione was found to be a sensitive electron acceptor of the reaction under aerobic as well as anaerobic conditions. The Km value of menadione for old yellow enzyme is as low as 2-3 x 10(-7) M in the presence or absence of superoxide dismutase. The rate enhancement of the cytochrome c reduction of old yellow enzyme with NADPH was about eight times in the presence of menadione. The rate increment was slightly higher under aerobic than anaerobic conditions. The rate enhancement by menadione enabled sensitive determination of the enzyme activity in the assay system, which contained NADPH, cytochrome c, menadione, and old yellow enzyme. In the reaction course, the semiquinone species of menadione was trapped by the reaction with t-butyl-alpha-phenylnitrone. The radical adduct was detected on EPR. The dyestuff, 2,6-dichlorophenolindophenol, was found to be reduced ineffectively even in the presence of menadione; moreover, it was inhibitory in the NADPH consumption reaction. Methylene blue or Lauth's violet, known to be capable of semiquinone formation, also behaved, like menadione, as a mediator of electron transport to cytochrome c. On the basis of the experimental results, the occurrence of the one electron transfer of the old yellow enzyme reaction was emphasized.
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PMID:Characterization of the electron acceptors of old yellow enzyme: mechanistic approach to the mode of one electron transfer from the enzyme to menadione or dyestuffs. 813 46


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