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)

Monodehydroascorbate reductase (EC 1.6.5.4) was purified from cucumber fruit to a homogeneous state as judged by polyacrylamide gel electrophoresis. The cucumber monodehydroascorbate reductase was a monomer with a molecular weight of 47,000. It contained 1 mol of FAD/mol of enzyme which was reduced by NAD(P)H and reoxidized by monodehydroascorbate. The enzyme had an exposed thiol group whose blockage with thiol reagents inhibited the electron transfer from NAD(P)H to the enzyme FAD. Both NADH and NADPH served as electron donors with Km values of 4.6 and 23 microM, respectively, and Vmax of 200 mol of NADH and 150 mol of NADPH oxidized mol of enzyme-1 s-1. The Km for monodehydroascorbate was 1.4 microM. The amino acid composition of the enzyme is presented. In addition to monodehydroascorbate, the enzyme catalyzed the reduction of ferricyanide and 2,6-dichloroindophenol but showed little reactivity with calf liver cytochrome b5 and horse heart cytochrome c. The kinetic data suggested a ping-pong mechanism for the monodehydroascorbate reductase-catalyzed reaction. Cucumber monodehydroascorbate reductase occurs in soluble form and can be distinguished from NADPH dehydrogenase, NADH dehydrogenase, DT diaphorase, microsome-bound NADH-cytochrome b5 reductase, and NADPH-cytochrome c reductase by its molecular weight, amino acid composition, and specificity of electron acceptors and donors.
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PMID:Monodehydroascorbate reductase from cucumber is a flavin adenine dinucleotide enzyme. 405 27

Transfer of mitochondrial protoheme to apocytochrome b5 in vitro was accomplished in a reconstitution system consisting of isolated mitochondria (donor) and apocytochrome b5 (acceptor), which required the existence of cytosol. Properties of formed cytochrome b5 were confirmed by its absorption spectra and the function as NADH-cytochrome c reductase. The content of formed cytochrome b5 was dependent on reaction time and the concentration of mitochondrial protoheme, apocytochrome b5, and cytosolic protein. This heme transfer protein was purified to homogeneity and identified with glutathione S-transferases (GSTs), by their same elution patterns in column chromatographies and the same degree of inhibited activities on the immunotitration study. Double immunodiffusion analysis revealed this protein to be GST-C2 (Yb' Yb'). These observations lead to the conclusion that Yb' subunit of GST located in the cytosol of rat liver stimulates the transfer of mitochondrial protoheme to apocytochrome b5, which indicates that GST has an unrecognized function as yet, involving on the biosynthesis of microsomal cytochrome b5.
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PMID:[Isolation and characterization of heme transfer protein involved in biosynthesis of microsomal cytochrome b5 from rat liver cytosol]. 407 16

Stearoyl-CoA desaturase activity in microsomes from lactating rat mammary gland is very low (0.05-0.15 nmol/min/mg of protein) regardless of lactating time. In such microsomes, reductase activities and content of cytochrome b5 are several-fold lower than in normal rat liver microsomes. Preincubation of the mammary microsomes with purified terminal desaturase gives a 55-fold stimulation of stearoyl-CoA desaturase activity, whereas preincubation with cytochrome b5 has no effect. However, preincubation of mammary microsomes with both cytochrome b5 and terminal desaturase results in a 200-fold stimulation of overall desaturation. These observations suggest that negligible stearoyl-CoA desaturase activity in lactating rat mammary microsomes is due to a cytochrome b5 content and the absence of terminal enzyme. The hepatic stearoyl-CoA desaturase activity increases 9-fold during lactation. There is little or no change in the NADH-cytochrome c reductase activity or in the concentrations of cytochrome b5 during this period, but the activity of the terminal desaturase increases with the increase of overall desaturation. These results suggest that liver is one of the more important sources of oleic acid for milk triglycerides.
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PMID:Stearoyl-coenzyme A desaturase activity in the mammary gland and liver of lactating rats. 612 66

Human placenta contains a thermostable, cytosolic NADH-diaphorase which is different from the other diaphorases and which we designate as diaphorase P. It is specific for NADH and reduces artificial substrates such as dichlorophenol and tetrazolium derivatives, but not natural substrates such as methemoglobin, cytochrome b5 or lipoate. It is antigenically distinct from the ubiquitous red-cell type NADH-diaphorase (soluble cytochrome b5 reductase) specified by the DIA1 locus. Using electrophoretic and immunologic methods, it was possible to detect diaphorase P in various fetal tissues (brain, liver, kidney, muscle), whereas was not found in adult tissues with the exception of the brain. This enzyme, the physiological role of which remains unknown, appears to belong, therefore, to the category of fetal proteins. Its resurgance in primary liver cancer was demonstrated in three cases.
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PMID:Diaphorase P: a new fetal isozyme identified in human placenta. 624 54

Peroxisomes were isolated from the livers of both control and clofibrate-treated rats. Two procedures, one with a sucrose gradient, and a second with Percoll gradients, were utilized. The Percoll procedure allowed contamination of the isolated peroxisome fraction on protein basis, by lysosomes (8%), by mitochondria (5%) and by microsomes (2%). The peroxisome fraction isolated by the sucrose gradient showed no significant contamination with mitochondria, but the fraction contained 13% microsomes. In addition to established peroxisomal enzymes, the isolated peroxisomes also contained cytochrome b5, NADH-cytochrome c reductase and NADPH-isocitrate dehydrogenase. The peroxisomal membranes were also separated from the content, and they were found to have a relatively high phospholipid/protein ratio (0.55).
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PMID:Isolation of peroxisomes from rat liver using sucrose and Percoll gradients. 627 64

n-Butyl and isoamyl alcohols decrease the steady-state fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene and enhance the efficiency of pyrene excimer formation when these probes are incorporated in rat-liver microsomal membrane, suggesting an increase in rotational and translational mobilities. Neither alcohol modifies NADH-ferricyanide reductase activity but both increase NADH-cytochrome c reductase activity. This was interpreted as an increase in the rate of lateral diffusion of the proteins cytochrome b5 and cytochrome b5 reductase as a consequence of the enhanced membrane lipid phase fluidity. Microsomal delta 9 and delta 6 desaturase activities in the presence of isoamyl alcohol were also studied. This alcohol decreases delta 9 desaturation when it is measured at a low substrate concentration (13 microM palmitic acid), but it is not modified when it is measured at a high substrate concentration (66 microM palmitic acid). delta 6 desaturation is diminished by isoamyl alcohol when it is measured with both 13 microM and 66 microM linoleic acid. The influence of isoamyl alcohol on the glucose-6-phosphatase system activity was also studied. In non-detergent-treated microsomes, isoamyl alcohol enhances glucose-6-phosphatase activity. However, if microsomes are previously treated with 0.1% Triton X-100 isoamyl alcohol does not modify this activity. The enhancement of the glucose 6-phosphate transport rate is not due to membrane permeability barrier disruption, since isoamyl alcohol does not modify mannose-6-phosphohydrolase latency. This would suggest that an increase in membrane lipid phase fluidity specifically activates glucose 6-phosphate transport across the membrane.
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PMID:Short-chain aliphatic alcohols increase rat-liver microsomal membrane fluidity and affect the activities of some microsomal membrane-bound enzymes. 631 21

Cells of the E3-24 mutant of the strain D273-10B of Saccharomyces cerevisiae, grown in a fermentable substrate not showing catabolite repression of respiration (2% galactose), are able to respire, in spite of their ubiquinone deficiency in mitochondrial membranes. Mitochondria isolated from these mutant cells oxidize exogenous NADH through a pathway insensitive to antimycin A but inhibited by cyanide. Addition of methanolic solutions of ubiquinone homologs stimulates the oxidation rate and restores antimycin A sensitivity in both isolated mitochondria and whole cells. Mersalyl preincubation of isolated mitochondria inhibits both NADH oxidation and NADH-cytochrome c oxido-reductase activity (assayed in the presence of cyanide) with the same pattern. Electrons resulting from the oxidation of exogenous NADH reduce both cytochrome b5 and endogenous cytochrome c. The increase in ionic strength stimulates NADH oxidation, which is also coupled to the ATP synthesis with an ATP/O ratio similar to that obtained with ascorbate plus N,N,N',N'-tetramethyl-p-phenylendiamine (TMPD) as substrate. The effect of cyanide on these activities and on NADH-induced endogenous cytochrome c reduction is also comparable. These results support the existence in vivo and in isolated mitochondria of a energy-conserving pathway for the oxidation of cytoplasmatic NADH not related to the dehydrogenases of the inner membrane, the ubiquinone, and the b-c1 complex, but involving a cytochrome c shuttle between the NADH-cytochrome c reductase of the outer membrane and cytochrome oxidase in the inner membrane.
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PMID:The oxidation of external NADH by an intermembrane electron transfer in mitochondria from the ubiquinone-deficient mutant E3-24 of Saccharomyces cerevisiae. 637 98

Rabbit lung and liver microsomes were subjected to three procedures which decreased NADPH cytochrome c reductase activity; flavoprotein antibody, trypsin and subtilisin digestion. The effects on benzphetamine and p-nitroanisole demethylation and amine metabolic-intermediate complex formation were investigated. In general, the proteolytic digestion had a greater inhibitory effect on oxidation reactions for a given loss of NADPH cytochrome c reductase activity than did flavoprotein antibody; and of the two proteases, subtilisin, which also diminishes the cytochrome b5 reduction pathway, had a greater inhibitory effect than trypsin. Subtilisin digestion had similar effects in both liver and lung microsomes; a loss of flavoprotein without a loss of cytochrome P-450; but whereas all three oxidative reactions decreased in unison as the flavoprotein was lost in the liver, benzphetamine demethylation was less susceptible to flavoprotein depletion than the other two reactions in lung microsomes. With trypsin digestion flavoprotein was removed without loss of cytochrome P-450 only in lung microsomes; in liver microsomes the cytochrome P-450 was susceptible to tryptic degradation. In lung microsomes, benzphetamine and p-nitroanisole demethylations were less susceptible to flavoprotein loss than metabolic-intermediate complex formation.
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PMID:The influence of in vitro procedures which diminish NADPH cytochrome c reductase activity on oxidative drug metabolism in lung and liver microsomes. 641 23

To clarify the molecular organization of NADH- and NADPH-dependent microsomal redox systems their isolated purified carriers were incorporated into immobilized azolectin layer with a higher viscosity than that of the liposomes. It was shown that the NADH-cytochrome c reductase activity characterizing the NADH-cytochrome b5 reductase and cytochrome b5 interaction sharply decreased in the immobilized system as compared to that in solution. However, the activity of hydroxylase reactions catalyzed by immobilized NADPH-cytochrome P-450 reductase and cytochrome P-450 was the same as in solution. This, the reconstitution in the immobilized phospholipid layer allowed to characterize NADH-cytochrome b5 reductase as a system operating on occasional collisions of its components. On the contrary, the diffusion of the NADPH-dependent redox chain carriers was not the rate-limiting step of the reaction.
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PMID:[Reconstitution of the monooxygenase system in a solution and in an immobilized phospholipid layer]. 642

The influence of ascorbate deficiency and megadosage on the metabolism of N-nitrosodimethylamine (NDMA) and N-nitrosodiethylamine (NDEA) was investigated in the guinea pig. After 21 days on a scorbutogenic diet, microsomal cytochrome P-450 and cytochrome b5 levels fell by 51 and 32%, respectively, while cytochrome c reductase activity remained constant. The activities of NDMA and NDEA dealkylase I were also depressed significantly. The Vmax of NDMA demethylase I and NDEA deethylase I was significantly depressed. Also, ascorbate deficiency significantly decreased the plasma clearance of both nitrosamines though the LD50 of neither were altered by ascorbate nutrition. Covalent binding of 14C from [14C]NDMA and [14C]NDEA to DNA obtained from liver slices was significantly lower in the deficient than in the control samples; megadosage appeared to have the opposite effect.
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PMID:The effects of ascorbic acid deficiency and excess on the metabolism and toxicity of N-nitrosodimethylamine and N-nitrosodiethylamine in the guinea pig. 642 11


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