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)

A histochemical analysis of reaction rates of a series of enzymes was performed in electromotor neurons of the weakly electric fish Apteronotus leptorhynchus. These neurons were selected because of their functional homogeneity. The high metabolic activity of these cells as well as their large size facilitate cytophotometric analysis in cryostat sections. Sections were incubated for the activity of hexokinase, glucose-6-phosphate dehydrogenase, succinate dehydrogenase, NADPH dehydrogenase, NADPH ferrihaemoprotein reductase and beta-hydroxybutyrate dehydrogenase. All media contained polyvinyl alcohol as tissue stabilizer and Nitro BT as final electron acceptor. Measurements were performed with a Vickers M85a cytophotometer. Linear relationships between the specific formation of formazan (test minus control reaction) and incubation time were obtained for all enzymes although some reactions showed an initial lag phase or an intercept with the ordinate. The relatively high activities of hexokinase, succinate dehydrogenase and the extremely low activity of hydroxybutyrate dehydrogenase indicate that energy is mainly supplied by glycolysis. Glucose-6-phosphate dehydrogenase showed a high activity whereas NADPH reductase and dehydrogenase activity were low in electromotor neurons, indicating that the NADPH generated is largely used for biosynthesis. Despite their synchronous firing pattern activity, electromotor neurons showed a considerable heterogeneity with respect to their metabolic activity.
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PMID:Enzyme reaction rate studies in electromotor neurons of the weakly electric fish Apteronotus leptorhynchus. 251 71

The metabolism of chemical carcinogens was investigated in liver preparations from 28 captive woodchucks (Marmota monax). Of these, 23 were naturally infected with the woodchuck hepatitis virus (WHV), and eight also had primary hepatocellular carcinoma (PHC). Twenty-nine parameters were investigated in liver subcellular fractions, including cross-reactivity with HBsAg, and biochemical parameters, such as gamma-glutamyl transpeptidase, cytochrome P-450 and microsomal monooxygenases (aryl hydrocarbon hydroxylase, ethoxycoumarin and ethoxyresorufin deethylases, aminopyrine and dimethylnitrosamine demethylases, and testosterone 7 alpha-, 16 alpha- and 6 beta-hydroxylases), uridine 5'-diphosphoglucuronosyl transferase, GSH and related enzymes (peroxidase, reductase and S-transferase), as well as other cytosolic enzyme activities (glucose 6-phosphate and 6-phosphogluconate dehydrogenases, NADPH- and NADH-dependent diaphorases, and DT diaphorase). In addition, liver preparations were used in order to quantify the metabolic activation into bacterial mutagens of five procarcinogens (aflatoxin B1, the pyrolysis products Trp-P-2 and MeIQ, 2-aminofluorene and dimethylnitrosamine) and the decrease of potency of three direct-acting mutagens (sodium dichromate, ICR 191 and 4-nitroquinoline 1-oxide). WHV infection produced a significant stimulation of carcinogen metabolism, as shown by the simultaneous change in detoxification parameters (GSH depletion) and activation indices (enhancement of microsomal monooxygenases and of procarcinogen activation into mutagenic metabolites). There were no significant differences between WHV-positive samples from animals without PHC and the noncancerous tissue of PHC-bearing animals, whereas a decrease of both activation and detoxification indices was recorded in the tumorous tissue. There was a considerable interindividual variability among WHV carriers, which was tentatively ascribed to genetic factors. Pregnancy was the only known factor influencing the results in WHV carriers. However, even by excluding pregnant animals, the effects on carcinogen metabolism produced by WHV infection were still statistically significant. These results, together with previous data obtained in humans, revealed that metabolic factors may play a role in the synergism between viral hepatitis and chemical hepatocarcinogens in the etiopathogenesis of PHC.
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PMID:Enhanced metabolic activation of chemical hepatocarcinogens in woodchucks infected with hepatitis B virus. 272 Sep 3

Spinach leaf ferredoxin and ferredoxin:NADP oxidoreductase as well as pig adrenodoxin and adrenodoxin reductase have been purified to homogeneity. Ferredoxin-NADP reductase and adrenodoxin-NADP reductase can perform the same diaphorase reactions (dichloroindophenol, ferricyanide and cytochrome c reduction) albeit not with the same efficiency. Despite the differences in their redox potentials, animal and plant ferredoxins can be used as heterologous substrates by the ferredoxin-NADP reductases from both sources. In heterologous systems, however, the ferredoxin/adrenodoxin concentrations must be increased approximately 100-fold in order to reach rates similar to those obtained in homologous systems. Ferredoxin and adrenodoxin can form complexes with the heterologous reductases as demonstrated by binding experiments on ferredoxin-Sepharose or ferredoxin-NADP-reductase-Sepharose and by the realization of difference spectra. Adrenodoxin also weakly substitutes for ferredoxin in NADP photoreduction, and can be used as an electron carrier in the light activation of the chloroplastic enzyme NADP-dependent malate dehydrogenase. In addition adrenodoxin is a good catalyst of pseudocyclic photophosphorylation, but not of cyclic phosphorylation and can serve as a substrate of glutamate synthase. These results are discussed with respect to the known structures of plant and animals ferredoxins and their respective reductases.
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PMID:On the specificity of pig adrenal ferredoxin (adrenodoxin) and spinach ferredoxin in electron-transfer reactions. 283 37

Biosynthesis of ferredoxin-NADP+ reductase in higher plants was investigated in relation with the mechanism of formation of the holoenzyme. The putative precursor of the flavoprotein, obtained after cell-free translation on a wheat germ extract primed with poly(A)-rich mRNA, was able to spontaneously bind free FAD, rendering a functional prereductase. The newly synthesized preholoenzyme showed diaphorase and cytochrome c reductase activities, an apparent molecular mass of 45 kDa, and contained FAD as the only flavin cofactor. It gave a positive reaction towards antisera against mature ferredoxin-NADP+ reductase. On the other hand, intracellular distribution of flavin-synthesizing enzymes indicates that FAD formation occurs in the cytoplasm; that is, in the same compartment as the site of reductase synthesis. On the basis of the preceding data a model is presented for the biosynthesis of the enzyme in vivo, involving conjugation of the apoprotein with FAD in the cytoplasm, followed by transport of the preholoreductase across the chloroplast envelope to reach its final destiny in the thylakoid membrane.
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PMID:Biosynthesis of ferredoxin-NADP+ oxidoreductase. Evidence for the formation of a functional preholoenzyme in the cytoplasmic compartment. 286 41

DT diaphorase [NAD(P)H:quinone oxidoreductase] activity was measured in subcellular fractions from homogenates of striatum, frontal cortex, hippocampus, cerebellum, hypothalamus and substantia nigra. This flavoprotein, which by definition oxidizes dihydronicotinamide adenine dinucleotide and dihydronicotinamide adenine dinucleotide phosphate at equal rates and is completely inhibited by 10(-5) M dicoumarol, was found to constitute 80-90% of the total dihydronicotinamide adenine dinucleotide- and dihydronicotinamide adenine dinucleotide phosphate-reductase activities in all brain regions studied. Antibodies raised against purified cytosolic DT diaphorase from the rat liver cross-reacted with the brain enzyme and inhibited soluble DT diaphorase from striatum and cerebellum to 80-90%. Immunohistochemical studies with the same antibodies demonstrated the occurrence of DT diaphorase immunoreactivity in a population of neurons in the substantia nigra and ventral tegmental area. In some neurons there was a colocalization of DT diaphorase and tyrosine hydroxylase-like immunoreactivity. The dense network of DT diaphorase-immunoreactive fibres in the striatum disappeared along with the dopaminergic innervation after 6-hydroxydopamine lesion. DT diaphorase immunoreactivity was also found in Bergmann glia, astrocytes and tanycytes. No correlation appeared to exist between the localization of neuronal DT diaphorase immunoreactivity and the dihydronicotinamide adenine dinucleotide phosphate-diaphorase-like activity, as defined by tetrazolium salt staining, used as a marker for certain peptidergic and cholinergic neurons. However, in, for example, glial cells in the cerebellum, DT diaphorase might contribute or be responsible for the histochemical dihydronicotinamide adenine dinucleotide phosphate-diaphorase activity.
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PMID:Distribution of DT diaphorase in the rat brain: biochemical and immunohistochemical studies. 290 55

NADH-cytochrome b5 reductase is the predominant NADH-diaphorase found in the human neutrophil (Blood 62:152, 1983). Although this reductase segregates with the light membranes of nitrogen-cavitated neutrophils separated on Percoll gradients (which include the plasma membrane markers alkaline phosphatase and NADPH-oxidase), it is approximately 95% excluded from plasma membrane-enriched phagocytic vacuoles. The reductase constitutes approximately 5% of the light membrane fraction FAD-flavoprotein (14.8 +/- 5.5 pmol/mg protein) and was found in equimolar concentration with a high potential b cytochrome also present in this light membrane fraction and tentatively identified as cytochrome b5. Isolation of the reductase from human neutrophils was accomplished by Triton X-114 solubilization of the light Percoll gradient membranes, followed by temperature-dependent phase separation and then affinity chromatography on AMP-Sepharose. The active preparation contained 1.3 mol FAD/mol protein, migrated on sodium dodecyl sulfate-polyacrylamide gels as a single band corresponding to an apparent mol wt of 45,000 daltons, exhibited a pl of 5.7 on chromatofocusing and was obtained in greater than 70% yield, with an overall purification of almost 900-fold. The purified enzyme was characterized by a high specificity for NADH as electron donor (Km = 6.4 mumol/L v Km greater than 1.6 mmol/L for NADPH) and exhibited a maximal turnover of ca. 30,000 min-1 at 22 degrees C with either ferricyanide or cytochrome b5 (Km = 10 nmol/L) as electron acceptor. Although the physical characterization and biochemical properties described here demonstrate that this neutrophil NADH b5 reductase is similar to the corresponding liver and erythrocyte enzymes, its unique function in the neutrophil has yet to be determined.
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PMID:Purification and characterization of the human neutrophil NADH-cytochrome b5 reductase. 299 39

Ferredoxin-NADP reductase from Euglena gracilis Klebs var. Bacillaris Cori purified to apparent homogeneity, yields a typical 36 kDa and an unusual 15 kDa polypeptide on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, exhibits a typical flavoprotein spectrum, contains FAD, and catalyzes NADPH-dependent iodonitrotetrazolium-violet diaphorase, NADPH-specific ferredoxin-dependent cytochrome-c-550 reductase and NADPH-NAD transhydrogenase activities. Rabbit antibody to the purified FNR blocks these activities specifically and also blocks the iodonitrotetrazolium-violet diaphorase activity of Euglena chloroplast completely. The low iodonitrotetrazolium-violet diaphorase activity in the plastidless mutant, W10BSmL, is mitochondrial and is not specifically blocked by the ferredoxin-NADP reductase antibody. Dark-grown non-dividing (resting) wild-type Euglena cells show a 4-fold increase in ferredoxin-NADP reductase activity during greening at 970 lx. Half of the low ferredoxin-NADP reductase activity in dark-grown cells is initially soluble, but by the end of chloroplast development nearly all of the enzyme is membrane-bound. The binding of ferredoxin-NADP reductase on exposure to light correlates with the extent of thylakoid membrane formation. Immunoblots of wild-type extracts during greening indicate that the 15 kDa polypeptide increases in the same manner as the extent of reductase binding to thylakoid membranes.
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PMID:Purification, properties, and cellular localization of Euglena ferredoxin-NADP reductase. 312 Jul 72

Yeast glutathione reductase exists in a single molecular form which exhibits preferred NADPH and weak NADH linked multifunctional activities. Kinetic parameters for the NADPH and NADH linked reductase, transhydrogenase, electron transferase and diaphorase reactions have been determined. The functional preference for the NADPH linked reductase reaction is kinetically related to the high catalytic efficiency and low dissociation constants for substrates. NADP+ and NAD+ may interact with two different sites or different kinetic forms of the enzyme. The active site disulfide and histidine are required for the reductase activity but are not essential to the transhydrogenase, electron transferase and diaphorase activities. Amidation of carboxyl groups and Co(II) chelation of glutathione reductase facilitate the electron transferase reaction presumably by encouraging the formation of an anionic flavosemiquinone.
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PMID:Multifunctional activities of yeast glutathione reductase. 329 44

Through the use of drug-adapted tissue culture cells, correlations have been observed between the level of specific enzymes and drug resistance. Drug resistance, however, may be due to multiple factors. To test whether the activity of daunorubicin reductase or NADPH diaphorase independently influences in vitro daunorubicin-induced cytotoxicity, we developed somatic cell hybrid clones to partially isolate these factors. This was accomplished by fusing daunorubicin-resistant myeloblast cells obtained from a patient with monosomy 7 leukemia to a daunorubicin-sensitive Chinese hamster cell line. The in vitro cytotoxicity of daunorubicin was compared in hybrid clones having variable enzyme activities; the concentrations of daunorubicin that inhibited the growth of clones by 50% did not differ by more than 2-fold, whereas daunorubicin reductase activities and NADPH diaphorase isozyme activities differed by more than 100- and 15-fold, respectively. These large differences in enzymatic activity were obtained in part by the suppression of specific hamster genes, indicating a regulatory control mechanism for xenobiotic enzymes. Our findings suggest that in this system substantial intercellular variation in the activity of these xenobiotic enzymes does not independently influence cellular resistance to daunorubicin.
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PMID:Use of somatic cell hybrids to analyze role of specific enzymes in daunorubicin cytotoxicity. 354 57

N-acetylcysteine (NAC) is often administered to respiratory patients with histories of exposure to noxious agents (e.g. cigarette smoke and atmospheric pollutants), which are known to act as glutathione (GSH) depletors and as cancer initiators and/or promoters. Since NAC is a precursor of intracellular GSH, we investigated its effects on GSH metabolism and on the biotransformation of carcinogenic and/or mutagenic compounds. In vitro, NAC induced a significant increase in oxidized glutathione (GSSG) reductase activity in rat liver preparations and counteracted the mutagenicity of direct-acting compounds (such as epichlorohydrin, hydrogen peroxide, 4-nitroquinoline-N-oxide and dichromate), as a result of its reducing and scavenging properties. At high concentrations, the drug completely inhibited the mutagenicity of procarcinogens (cigarette smoke condensate, tryptophan pyrolysate, cyclophosphamide, 2-aminofluorene, benzo(a)pyrene and aflatoxin B1) by binding their electrophilic metabolites. In contrast, their metabolic activation was stimulated by decreasing NAC concentrations, especially when liver preparations from enzyme-induced rats were used. Lung and liver subcellular preparations of rats treated in vivo with NAC, in various combinations with enzyme inducers and/or GSH depletors, also affected the mutagenicity of a number of compounds. NAC generally increased intracellular GSH and restored its levels following depletion. It did not affect the levels nor the spectral properties of cytochromes P-450 in pulmonary and hepatic microsomes, whereas it stimulated, especially in Aroclor-pretreated animals, cytosolic enzyme activities involved in NADP or GSSG reduction (G6PD, 6PGD and GSSG reductase) and in the reductive detoxification of xenobiotics (DT diaphorase). When administered with the diet, at a nontoxic posology (120 mg/kg b.w.), NAC markedly inhibited the induction of lung tumors in mice by a potent carcinogen (urethane).
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PMID:Metabolic, desmutagenic and anticarcinogenic effects of N-acetylcysteine. 380 42


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