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)

22 revertants of Saccharomyces cerevisiae with intragenic suppressors (supa) of cob exon mutations (G. Burger, Mol. Gen. Genet., in the press) were analyzed. They display either a reduced amount of cytochrome b, or a shifted maximum absorption wavelength of total cytochrome b or a reduced growth rate on glycerol. The relationship of physico-chemical properties (content, light absorption and midpoint potential of cytochromes bK and bT) and functional properties (electron transport and energy yield) has been examined. In seven of eight revertants with a shifted maximum absorption wavelength of cytochrome b neither growth rate nor electron transfer activity was affected. In 13 of 14 revertants, reduced content of cytochrome b corresponds to a reduced electron transport rate through the cytochrome bc1 segment. A lower enzymatic activity, which is not due to a quantitative but to a qualitative alteration of cytochrome b was found in two revertants. Two revertants show electron transport rates of wild-type level concomitant with a reduced growth rate on glycerol, obviously due to a less efficient energy coupling. All revertants were shown to contain a high and a low potential cytochrome b, referred to as bK and bT. Those cob-/supa mutations which shift the maximum absorption wavelength or diminish the content of cytochrome b affect both b cytochromes in all cases. The results support that electron transport and energy conservation are catalyzed by the unity of cytochrome bK and bT and that both heme centers are bound to an identical apoenzyme. Comparing electron flow rates of succinate:cytochrome c oxidoreductase and NADH:cytochrome c oxidoreductase in cob- mutants and two revertants provides evidence that ubiquinone does not constitute a homogeneous pool, suggested by the dissimilar interaction of both dehydrogenases with the bc1 segment.
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PMID:Cytochrome b of cob revertants in yeast. Bioenergetic characterization of revertants with reduced content and shifted maximum absorption wavelength of cytochrome b. 608 48

It was shown that malate dehydrogenase of isolated membranes of the gramicidin S producer Bacillus brevis var. G.-B. (R.-form) is completely inhibited by the antibiotic (approximately 200 mkg/mg of protein). Succinate and NADH dehydrogenases at concentration up to 1 mg per mg of protein are insensitive to it, while corresponding oxidases are inhibited by the antibiotic not more than by 65 -- 75% apparently due to partial damage of the terminal parts of the respiratory chain. The respiration of the producer intact cells is inhibited by exogenous gramicidin S by not more than 55 -- 60%, while the respiration of antibiotic-sensitive cells of M.lysodeikticus is inhibited completely. It was shown that phosphatidyl ethanolamine (50%), phosphatidyl glycerol (15% and diphosphatidyl glycerol (25%) are the major phospholipid components of the membranes of the given strain of Bac. brevis. It was assumed that the resistance of Bac. brevis cells to gramicidin S is partly due to the constant ratio of the charged and amphoteric phospholipids. Using 31P-NMR spectroscopy, the kinetics of free phosphoric compounds in the cells and cell extracts of Bac. brevis during culture growth and gramicidin S synthesis were studied. The content of carbohydrate monophosphate, remained unaffected, while that of nucleoside di- and triphosphates and dinucleotides was low and at definite density and gramicidin S content (above 100 mkg/ml) fell down below the resolution capacity of the method employed. Evidence for gramicidin S localization of the Bac. brevis membrane and possible causes for the manifestation of the NADH dehydrogenase activity at a certain stage of culture growth are discussed.
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PMID:[Localization of gramicidin S on the cytoplasmic membrane of Bacillus brevis and its effect on the activity of membrane enzymes]. 616 75

Among 979 non-glycerol growers of the yeast Schizosaccharomyces pombe, 40 strains were found to be deficient in the mitochondrial ATPase activity. Three of them exhibited an alteration in either the alpha or beta subunits of the F1ATPase. The alpha subunit was not immunodetected in the A23/13 mutant. The beta subunit was not immuno-detected in the B59/1 mutant. The existence of these two mutants shows that the alpha and beta subunits can be present independently of each other in the inner mitochondrial membrane. The beta subunit of the mutant F25/28 had a slower electrophoretic mobility than that of the wild-type beta subunit. This phenotype indicates abnormal processing or specific modification of the beta subunit. All mutants showed reduced activities of the NADH-cytochrome c reductase and of the cytochrome oxidase and a decreased synthesis of cytochrome aa3 and cytochrome b. This pleiotropic phenotype appears to result from specific modifications in the mitochondrial protein synthesis. The mitochondrial synthesis of four polypeptides (three cytochrome oxidase and one cytochrome b subunits) was markedly decreased or absent while three new polypeptides (Mr = 54000, 20000 and 15000) were detected in all the mutants analysed. This observation suggests that a functional F1ATPase is necessary for the correct synthesis and/or assembly of the mitochondrially made components of the cytochrome oxidase and cytochrome b complexes.
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PMID:Alterations of the alpha or beta subunits of the mitochondrial ATPase in yeast mutants. 621 96

The role of phospholipids in ubiquinol-cytochrome c reductase has been studied by the following methods: (1) removal and restoration of phospholipids, (2) circular dichroism measurements, and (3) phospholipase A2 treatment. Over 90% of the phospholipids in the cytochrome b--c1 III complex (a highly purified ubiquinol-cytochrome c reductase) can be removed by repeated precipitation with ammonium sulfate in the presence of 0.5% sodium cholate. The delipidated enzyme complex is inactive. Full restoration of enzymatic activity can only be achieved with a freshly prepared delipidated enzyme complex, made in the presence of 20% glycerol. As the age of the delipidated enzyme increased, the amount of activity restored decreased and the incubation time required to reach maximal activity increased. Removal of phospholipids from the cytochrome b--c1 III complex resulted in an immediate decrease of approximately 15% in molar ellipticities in both the far-UV and the Soret regions. A further decrease in ellipticities was observed upon incubation of the delipidated enzyme at 0 degrees C in 50 mM phosphate buffer, pH 7.4. Replenishing phospholipids to the delipidated enzyme complex restored enzymatic activity and the molar ellipticity in both regions. The absolute requirement for phospholipids in the cytochrome b--c1 III complex was also demonstrated by treatment of the enzyme with purified phospholipase A2. The inactivation of the cytochrome b--c1 III complex by phospholipase A2 was not prevented by the presence of excess exogenous ubiquinone but was prevented by the presence of ethylenediaminetetracetic acid (EDTA). The enzymatic activity of the phospholipase A2 treated complex is fully restorable upon the addition of EDTA and phospholipids.
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PMID:Structural role of phospholipids in ubiquinol-cytochrome c reductase. 625 87

Synthesis of phosphatidylglycerol from CDPdiacylglycerol and glycerol 3-phosphate by membranous subcellular fractions of rat lung and liver was optimal when assayed in the presence of bovine serum albumin and Triton X-100. Specific activities of glycerolphosphate phosphatidyltransferase in all membranous subcellular fractions of lung were several times higher than the corresponding fractions from liver. Distribution of this enzyme in subcellular fractions of lung or liver closely parallel the activity of the mitochondrial enzymes monoamine oxidase and succinate cytochrome c reductase. The phosphatidylglycerol-synthesizing activity in microsomes of both lung and liver was a minor fraction of total tissue activity and could be interpreted as due either to contamination with outer mitochondrial membrane or to a small amount of activity innate to microsomes. These results suggest that phosphatidylglycerol, which is believed to be a component of pulmonary surfactant, is synthesized by lung at a rapid rate relative to liver and that the subcellular distribution of its synthesis is similar in both tissues, with mitochondria as the major site.
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PMID:Optimal assay and subcellular location of phosphatidylglycerol synthesis in lung. 626 66

We describe two fully enzymic methods, fluorometric and colorimetric, for determination of triglycerides (triacylglycerols) in serum. Samples are incubated with microbial lipase for 10 min, and the glycerol released from the triglycerides is oxidized by NAD+ in the presence of glycerol dehydrogenase. In the fluorometric method, the resulting NADH is in turn oxidized by resazurin as catalyzed by diaphorase to form resorufin, a highly fluorescent compound. In the colorimetric method, the NADH is oxidized by coupling with a tetrazolium salt/diaphorase system to form formazan, a highly colored compound. Calibration curves, constructed by plotting change in fluorescence or absorbance vs concentration of triglycerides, were linear up to 6 and 5 g of triglycerides per liter of serum for the fluorometric and colorimetric methods, respectively. The assays require only 5 and 15 microL of serum for fluorometry and colorimetry, respectively. The CV was 0.59% for the fluorometric method, 0.91% for the colorimetric procedure. The time for analysis for either method is less than 15 min. The results correlate well with those obtained by the Dow Diagnostic Kit method, a colorimetric method in which glycerol kinase and glycerol-1-phosphate dehydrogenase form NADH from ATP and NAD+ in the presence of glycerol and glycerol 1-phosphate.
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PMID:Fluorometric and colorimetric enzymic determination of triglycerides (triacylglycerols) in serum. 689 89

Spontaneous mutants resistant to vanadate, arsenate or thiophosphate were isolated from a haploid strain of Saccharomyces cerevisiae. These three anions have an inhibitory effect on some mitochondrial functions and at the level of glyceraldehyde 3-phosphate dehydrogenase, a glycolysis enzyme. All the selected mutants had the same phenotype: they were deficient in alcohol dehydrogenase I, the terminal enzyme of the glycolysis, and possessed a high content of cytochrome c oxidase, the terminal enzyme of the respiratory chain. Moreover, cytochrome c oxidase biosynthesis had become insensitive to the catabolite repression, while the biosynthesis of the other enzymes sensitive to this phenomenon were always inhibited by glucose. Metabolic effects of this pleiotropic mutation manifested themselves in the following ways. 1. Growth rate and final cell mass were enhanced, compared to the wild type, when cells were grown on glucose or on glycerol, but not on lactate or ethanol. 2. Growth under anaerobiosis was nil and mutants did not ferment. 3. Mitochondrial respiration of the mutant strains was identical to the wild type with succinate or 2-oxo-glutarate as substrate, and weak with ethanol. But with added NADH, respiration rate of the mutants was higher than that of the wild type and partially insensitive to antimycin, even when cells were grown in repression conditions. It is postulated that in mutants strains, NADH produced at the level of glyceraldehyde 3-phosphate dehydrogenase, failing to be reoxidized via alcohol dehydrogenase, could be reoxidized with a high turnover owing to the enhancement of the amount of cytochrome c oxidase. Since NADH reoxidation is partially insensitive to antimycin, a secondary pathway going from external NADH dehydrogenase to cytochrome c oxidase is suggested.
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PMID:New mutants resistant to glucose repression affected in the regulation of the NADH reoxidation. 704 95

There have been considerable interlaboratory variations in the reported levels of rat brain microsomal cytochrome P450 and associated monooxygenase activities. To ascertain if the variability could be accountable, at least in part, to different methodologies used for microsome preparation, cytochrome P450 monooxygenase components and activities were directly compared herein using brain microsome prepared by various methods. Rat brain microsome isolated using a calcium aggregation method in the presence of dithiothreitol and glycerol contained approximately 100 pmol of cytochrome P450/mg protein. Considerably lower cytochrome P450 levels (e.g. 20-40 pmol/mg protein) were found in brain microsome prepared in a more conventional manner using Tris or phosphate buffers without glycerol and dithiothreitol. The NADPH cytochrome c reductase activity was consistently approximately 23-25 nmol of cytochrome c reduced/min/mg protein, whatever the method of preparation of the brain microsome. Cytochrome P450-associated monooxygenase activities, namely morphine N-demethylase and ethoxycoumarin O-deethylase, were dependent on the amount of protein in the incubation medium, the length of incubation, and the ratio of the concentration of the substrate to the amount of protein in the incubation mixture. The specific activity of morphine N-demethylase was constant over a range of protein concentration, if the ratio of the concentration of the substrate to the protein was kept constant.
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PMID:Rat brain cytochrome P450. Reassessment of monooxygenase activities and cytochrome P450 levels. 758 47

The rate of transfer of reducing equivalents from cytoplasm to mitochondria has been examined in Ehrlich ascites tumour cells incubated in the presence of lactate. The flux of reducing equivalents was determined from the rate of metabolism of reduced intermediates that are oxidized within the cytosol. The magnitude of the flux of reducing equivalents was dependent on both the concentration of added lactate and the presence of carbohydrate. The rate of flux was twice as great in the presence of glucose and four times as high when glucose and lactate were added together as when lactate was the only added substrate. Fructose was less effective than glucose in stimulating reducing equivalent flux. In the presence of glucose or fructose, there was a substantial accumulation of hexose phosphates, dihydroxyacetone phosphate and glycerol 3-phosphate. Rotenone, an inhibitor of NADH dehydrogenase, and amino-oxyacetate, which inhibits the malate/aspartate shuttle, were powerful suppressors of reducing equivalent flux from lactate as sole substrate, but were much less potent in the presence of carbohydrate. Antimycin substantially inhibited reducing equivalent flux from all combinations of added substrates, consistent with its ability to block oxidation of reducing equivalents transferred by both the malate/aspartate and glycerol 3-phosphate shuttles. The glycerol 3-phosphate shuttle represents around 80% of the maximum total observed activity but is active only while glycolytic intermediates are present to provide the necessary substrates of the shuttle. This Ehrlich ascites cell line has an essentially similar total reducing equivalent shuttle capacity to that of isolated hepatocytes.
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PMID:Substrate-dependent utilization of the glycerol 3-phosphate or malate/aspartate redox shuttles by Ehrlich ascites cells. 765 9

Iron catalyzed free radical formation and lipid peroxidation are accepted mechanisms of heme protein-induced acute renal failure. However, the source(s) of those free radicals which trigger lipid peroxidation in proximal tubular cells remains unknown. This study tested the potential involvement of mitochondrial electron transport, xanthine oxidase activity, and arachidonic acid metabolism in the heme-induced peroxidative state. The impact of cytosolic Ca2+ loading also was assessed. Rhabdomyolysis was induced in mice by glycerol injection, and two hours later heme-laden proximal tubular segments (PTS) were isolated for study. PTS from normal mice served as controls. During 30 to 60 minute incubations, heme loaded PTS developed progressive cytotoxicity (LDH release) and iron-dependent lipid peroxidation (malondialdehyde, MDA, generation; inhibited by deferoxamine). Site 2 (antimycin A) or site 3 (cyanide, hypoxia) mitochondrial respiratory chain inhibition completely blocked lipid peroxidation, whereas site 1 inhibition (rotenone) doubled its extent (presumably by shunting NADH through NADH dehydrogenase, a free radical generating system). Conversely, these agents did not substantially alter MDA in normal PTS. Normal and heme loaded PTS developed comparable degrees of LDH release during respiratory blockade irrespective of increased or decreased MDA production (indicating that lipid peroxidation was not a critical determinant of cell death). Neither increasing free arachidonic acid (PLA2 treatment) nor adding cyclooxygenase/lipoxygenase/cytochrome p450 inhibitors conferred a consistent protective effect. Altering free Ca2+ status (chelators; ionophore addition) and xanthine oxidase inhibition had no discernible impacts. Despite mitochondrial free radical production, mitochondrial function, as assessed by the ATP/ADP ratio, seemingly remained intact. In conclusion, (1) the terminal mitochondrial respiratory chain is the dominant source of free radicals which trigger PTS lipid peroxidation; (2) iron is a required secondary factor; (3) although mitochondria fuel lipid peroxidation, they do not appear to be critical targets of the heme-induced oxidant attack.
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PMID:Mitochondrial free radical production induces lipid peroxidation during myohemoglobinuria. 864 15


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