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)

Neutrophil myeloperoxidase, hydrogen peroxide, and chloride constitute a potent antimicrobial system with multiple effects on microbial cytoplasmic membranes. Among these is inhibition of succinate-dependent respiration mediated, principally, through inactivation of succinate dehydrogenase. Succinate-dependent respiration is inhibited at rates that correlate with loss of microbial viability, suggesting that loss of respiration might contribute to the microbicidal event. Because respiration in Escherichia coli can be mediated by dehydrogenases other than succinate dehydrogenase, the effects of the myeloperoxidase system on other membrane dehydrogenases were evaluated by histochemical activity stains of electrophoretically separated membrane proteins. Two bands of succinate dehydrogenase activity proved the most susceptible to inactivation with complete loss of staining activity within 20 min, under the conditions employed. A group with intermediate susceptibility, consisting of lactate, malate, glycerol-3-phosphate, and dihydroorotate dehydrogenases as well as three bands of glucose-6-phosphate dehydrogenase, was almost completely inactivated within 30 min. The relatively resistant group, including the dehydrogenases for glutamate, NADH, and NADPH and the remaining bands of glucose-6-phosphate dehydrogenase, retained substantial amounts of diaphorase activity for up to 60 min of incubation with the myeloperoxidase system. The differential effects of myeloperoxidase on dehydrogenase inactivation could not be correlated with published enzyme contents of flavin or iron-sulfur centers, potential targets of myeloperoxidase-derived oxidants. Despite the relative resistance of NADH dehydrogenase/diaphorase activity to myeloperoxidase-mediated inactivation, electron transport particles prepared from E. coli incubated for 20 min with the myeloperoxidase system lost 55% of their NADH oxidase activity.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Differential inactivation of Escherichia coli membrane dehydrogenases by a myeloperoxidase-mediated antimicrobial system. 169 36

1. After immunization of BALB/c mouse, four monoclonal antibodies against soluble NADH diaphorase from ejaculated boar spermatozoa were produced and characterized. The monoclonal antibodies were designated as follows Mab 1F2, Mab 4E2, Mab 5B8, Mab 5D8. 2. These monoclonal antibodies react with other enzyme forms-sedimentary NADH and NADPH and soluble NADPH and inhibit (although not completely) their activity. It is supposed that different forms of the enzyme share some common epitopes. 3. Treatment of ejaculated boar semen with 2O-methylcholanthrene causes an increase of the activity of the soluble diaphorase form only. 4. These results lead to the assumption that the sperm diaphorase is a dynamic enzyme system consisting of four immunologically similar isoenzymes although their functions are different.
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PMID:Characterization of NAD(P)H diaphorase from boar spermatozoa using specific monoclonal antibodies. 170 1

Specimens of the seawater fish annular seabream (Diplodus annularis) were caught from a polluted harbor area and from a clean reference area. Seawater concentrates and fish-muscle extracts were not mutagenic in the Salmonella reversion test. Liver preparations of fish from the 2 sources were comparatively assayed for microsomal mixed-function oxidases and cytosolic biochemical parameters, as well as for the ability of S12 fractions to activate promutagens or to detoxify direct-acting mutagens. A shift of the cytochrome P-450 peak from 450.3 to 448.5 was accompanied by a 4.5-fold increase in arylhydrocarbon hydroxylase activity in fish living in the polluted environment. At the same time, glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase were doubled in the cytosol of the same animals, while reduced glutathione (GSH) peroxidase and GSH S-transferase were slightly yet significantly depressed. No significant difference was recorded for other biochemical parameters, including GSH, oxidized glutathione (GSSG) reductase, NADH- and NADPH-dependent diaphorases, and DT diaphorase. In parallel, fish exposed to polluted seawater exhibited a significant and marked enhancement of the metabolic activation of the pyrolysis product Trp-P-2 and of benzo[a]pyrene-trans-7,8-diol, and at the same time were less efficient in detoxifying the antitumor compound ICR 191. Liver S12 fractions from both sources efficiently decreased the direct mutagenicity of sodium dichromate, and failed to activate benzo[a]pyrene and aflatoxin B1 to mutagenic metabolites. These results provide evidence that both biochemical parameters and the overall capacity of fish liver to activate or detoxify certain mutagens can be assumed to be sensitive indicators of exposure to mixed organic pollutants in the marine environment.
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PMID:Enhanced liver metabolism of mutagens and carcinogens in fish living in polluted seawater. 170 59

NADPH diaphorase histochemistry selectively labels a number of discrete populations of neurons throughout the nervous system. This simple and robust technique has been used in a great many experimental and neuropathological studies; however, the function of this enzyme has remained a matter of speculation. We, therefore, undertook to characterize this enzyme biochemically. With biochemical and immunochemical assays, NADPH diaphorase was purified to apparent homogeneity from rat brain by affinity chromatography and anion-exchange HPLC. Western (immunoblot) transfer and immunostaining with an antibody specific for NADPH diaphorase labeled a single protein of 150 kDa. Nitric oxide synthase was recently shown to be a 150-kDa, NADPH-dependent enzyme in brain. It is responsible for the calcium/calmodulin-dependent synthesis of the guanylyl cyclase activator nitric oxide from L-arginine. We have found that nitric oxide synthase activity and NADPH diaphorase copurify to homogeneity and that both activities could be immunoprecipitated with an antibody recognizing neuronal NADPH diaphorase. Furthermore, nitric oxide synthase was competitively inhibited by the NADPH diaphorase substrate, nitro blue tetrazolium. Thus, neuronal NADPH diaphorase is a nitric oxide synthase, and NADPH diaphorase histochemistry, therefore, provides a specific histochemical marker for neurons producing nitric oxide.
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PMID:Neuronal NADPH diaphorase is a nitric oxide synthase. 170 73

NADPH diaphorase staining neurons, uniquely resistant to toxic insults and neurodegenerative disorders, have been colocalized with neurons in the brain and peripheral tissue containing nitric oxide synthase (EC 1.14.23.-), which generates nitric oxide (NO), a recently identified neuronal messenger molecule. In the corpus striatum and cerebral cortex, NO synthase immunoreactivity and NADPH diaphorase staining are colocalized in medium to large aspiny neurons. These same neurons colocalize with somatostatin and neuropeptide Y immunoreactivity. NO synthase immunoreactivity and NADPH diaphorase staining are colocalized in the pedunculopontine nucleus with choline acetyltransferase-containing cells and are also colocalized in amacrine cells of the inner nuclear layer and ganglion cells of the retina, myenteric plexus neurons of the intestine, and ganglion cells of the adrenal medulla. Transfection of human kidney cells with NO synthase cDNA elicits NADPH diaphorase staining. The ratio of NO synthase to NADPH diaphorase staining in the transfected cells is the same as in neurons, indicating that NO synthase fully accounts for observed NADPH staining. The identity of neuronal NO synthase and NADPH diaphorase suggests a role for NO in modulating neurotoxicity.
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PMID:Nitric oxide synthase and neuronal NADPH diaphorase are identical in brain and peripheral tissues. 171 81

Lipoamide dehydrogenase (E.C. 1.6.4.3) was found in Trypanosoma cruzi, Tulahuen strain, stocks Tul-2 and Q501, and CA-1 strain. After differential centrifugation of epimastigote homogenates, ammonium sulfate fractionation of the 105,000 g supernatant yielded a partially purified preparation which precipitated between 0.40 and 0.80 ammonium sulfate saturation. The enzyme (a) catalyzed the oxidation of dihydrolipoamide by NAD+ and the reduction of lipoamide by NADH, the forward reaction being 2.5-fold faster than the reverse reaction; (b) exhibited hyperbolic dependence on substrate concentration and (c) possessed diaphorase activity which was less than 5% of the lipoamide reductase activity. The NADH-reduced enzyme was inhibited by arsenite, cadmium and p-chloromercuribenzoate in a concentration-dependent manner. Substrate specificity allowed lipoamide dehydrogenase to be differentiated from T. cruzi trypanothione reductase and other NADPH-dependent flavoenzymes. After cell disruption, lipoamide dehydrogenase was found mostly in the cytosolic fraction and no evidence for association with the plasma membrane was obtained.
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PMID:Lipoamide dehydrogenase from Trypanosoma cruzi: some properties and cellular localization. 176 55

An NADPH-dependent membrane-bound flavoprotein dehydrogenase, assayed as a catalyst of electron transfer from NADPH to cytochrome c, was extracted from membranes of rabbit peritoneal neutrophils with Triton X-100 and sodium deoxycholate in the presence of diisopropylfluorophosphate as antiprotease, and purified to electrophoretic homogeneity. The purified enzyme in detergent was able to enhance the rate of formation of the superoxide anion O2- in a cell-free system, consisting of membrane and cytosolic fractions from resting neutrophils complemented with arachidonic acid, guanosine 5'-[gamma- thio]triphosphate and Mg2+. This suggested that the NADPH dehydrogenase was a component of the rabbit neutrophil oxidase complex. The purification factor of the enzyme with respect to the membrane fraction was close to 1000 and the recovery of activity was 33%. FMN and FAD were associated with the enzyme in a molar ratio close to 1. On SDS/PAGE, the enzyme migrated with a molecular mass of 77 kDa. A similar mass was determined by filtration on a molecular sieve. The isoelectric point of this enzyme was 4.7 +/- 0.1. Its activity was maximal between pH 7.5 and pH 8.5, and depended on the ionic strength of the medium, with a maximum at an ionic strength of 0.5. Reduction of cytochrome c by NADPH obeyed Michaelis-Menten kinetics with a KM value of 15 microM for cytochrome c. When NADPH was the variable substrate, a KM value of 1.9 microM for NADPH was found, but a significant deviation from Michaelis-Menten kinetics was observed at high concentrations of NADPH. Mersalyl strongly inhibited the reductase activity when added to the enzyme prior to NADPH; preincubation of the enzyme with NADPH considerably reduced the inhibitory efficiency of mersalyl. A partially proteolyzed water-soluble, active, form of enzyme with a molecular mass of 67 kDa was prepared. The proteolyzed enzyme exhibited the same specificity, and kinetic behavior with respect to NADPH, and the same dependency on the ionic strength, as the native enzyme.
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PMID:NADPH-cytochrome c reductase from rabbit peritoneal neutrophils. Purification, properties and function in the respiratory burst. 184 86

NAD(P)H: quinone oxidoreductase (NQO1) is believed to be protective against cancer and toxicity caused by exposure to quinones and their metabolic precursors. This enzyme catalyzes the two-electron reduction of compounds, compared with one-electron reduction mediated by NADPH: cytochrome-P450 oxidoreductase which produces toxic and mutagenic free radicals. Recently we cloned and sequenced the cDNA encoding human 2.3,7,8-tetrachlorodibenzo-p-dioxin (dioxin)-inducible cytosolic NQO1 [Jaiswal et al. (1988) J. Biol. Chem. 263, 13572-13578] and provided preliminary evidence that this enzyme may correspond to diaphorase 4, an enzymatic activity present in various tissues that catalyzes the reduction of a variety of quinones by both NADH and NADPH [Edwards et al. (1980) Biochem. J. 187, 429-436]. In the present report we characterize the catalytic properties of the protein encoded by the NQO1 cDNA. The enzyme was synthesized in monkey kidney COS-1 cells transfected with a pMT2-based expression plasmid containing the NQO1 cDNA. Western blot analysis of the transfected cells using an antibody against rat liver cytosolic NQO1 revealed a 31-kDa band that was not detected in nontransfected cells. This band corresponded to a polypeptide with the same electrophoretic mobility as the endogenous NQO1 protein detected in the human hepatoblastoma (Hep-G2) cells with the same antibody. The immunoreactive protein detected in human Hep-G2 cells was induced approximately fourfold by exposure of the cultures to dioxin, an increase commensurate with the increased in quinone oxidoreductase activity. These studies suggest that the protein encoded by NQO1 cDNA is indeed similar, if not identical, to the dioxin-inducible protein band detected in human Hep-G2 cells. Further characterization of the product of NQO1 cDNA, which was present at approximately 20-30-fold higher levels in transfected COS cells than the endogenous product in uninduced human Hep-G2 cells indicated that it had very high capacity (greater than 1000-fold over background) to catalyze the reduction of 2.6-dichloroindophenol and menadione. Besides these two commonly used substrates for quinone reductase, the expressed NQO1 protein also effectively metabolized 2,6-dimethylbenzoquinone, methylene blue, p-benzoquinone, 1,4-naphthoquinone, 2-methyl-1,4-benzoquinone, with the latter being the most potent electron acceptor at 50 microM concentration of the substrate.
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PMID:The human dioxin-inducible NAD(P)H: quinone oxidoreductase cDNA-encoded protein expressed in COS-1 cells is identical to diaphorase 4. 189 80

The activity of ferredoxin: NADP+ reductase (FNR) was found to decline to approximately 20% maximal levels with little or no loss in enzyme levels when cultures of the cyanobacterium Anabaena variabilis were maintained in the stationary phase of growth. Re-activation of enzyme activity occurred when cells were diluted into either fresh or re-utilized media and illuminated. This reversible de-activation/re-activation process was found, in vivo, to be dependent on the intensity of light illuminating the cells. The de-activated form of FNR was purified to homogeneity and exhibited the same molecular mass, isoelectric-focusing pattern and N-terminal amino acid sequence as the native form. Both de-activated and native FNR preparations each exhibited three reactive thiol groups on denaturation in urea; however, the rate of reaction with Ellman's reagent was much faster with the de-activated form than with the native form. Both preparations contain a single disulphide bond. Upon reduction of the disulphide bond in either form of the enzyme, the five reactive thiol groups exhibited identical reactivities in the presence of urea. Steady-state kinetic analysis of the de-activated form showed a marked increase in Km values for NADPH in diaphorase assays and an increase in Km for ferredoxin in the ferredoxin-mediated reduction of cytochrome c. No significant difference in kcat. was observed in comparison of the de-activated with the native form in any of the above assays; however, the de-activated form did exhibit a lower kcat. value in the transhydrogenase assay. The de-activated form of FNR bound ferredoxin with a 16-fold lower affinity than the native enzyme. These data suggest that the de-activation of FNR in vivo in response to low light intensity involves an alteration in protein structure, possibly via an intramolecular thiol disulphide interchange, which influences the interaction of the enzyme with its substrates.
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PMID:Light-dependent de-activation/re-activation of Anabaena variabilis ferredoxin: NADP+ reductase. 190 89

Pyruvate:NADP+ oxidoreductase from Euglena gracilis, a homodimeric protein with a molecular weight of 309 kDa, is an iron-sulfur flavoenzyme that contains thiamin pyrophosphate (TPP). The functional structure of the enzyme was studied by a limited proteolysis experiment using trypsin. The evidence obtained shows that the enzyme consists of two functional domains, one of which contains an iron-sulfur cluster, which can be isolated as a homodimeric fragment of approximately 220 kDa by proteolysis. The other domain that contains FAD is released as a monomeric fragment of approximately 55 kDa. The pyruvate dehydrogenase reaction is still catalyzed by the large fragment when NADP+ is substituted by methyl viologen, while the small fragment retains a diaphorase-like electron-transfer activity from NADPH to MV. It is thus shown that pyruvate is oxidized in a CoA-dependent reaction to form CO2 and acetyl-CoA in the iron-sulfur domain, and that the two electrons formed are transferred to the FAD domain in which NADP+ is reduced. TPP is considered to be associated in the iron-sulfur domain. The NH2-terminal sequences of the enzyme and its proteolytic fragments reveal that the iron-sulfur domain occurs in the NH2-terminal side of the enzyme. For elucidation of the O2 instability of the enzyme, limited proteolysis was attempted in air. The tryptic fragment derived from the iron-sulfur domain, similar to the native enzyme, appears to be inactivated by direct contact with O2. In contrast, the FAD domain, when separated from the other domain, is quite stable in air, although the diaphorase activity decays when the native enzyme is exposed to O2.
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PMID:Pyruvate:NADP+ oxidoreductase from Euglena gracilis: limited proteolysis of the enzyme with trypsin. 191 Feb 87


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