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)

Xanthine dehydrogenase (XDH) from the unicellular green alga Chlamydomonas reinhardtii has been purified to electrophoretic homogeneity by a procedure which includes several conventional steps (gel filtration, anion exchange chromatography and preparative gel electrophoresis). The purified protein exhibited a specific activity of 5.7 units/mg protein (turnover number = 1.9 .10(3) min-1) and a remarkable instability at room temperature. Spectral properties were identical to those reported for other xanthine-oxidizing enzymes with absorption maxima in the 420-450 nm region and a shoulder at 556 nm characteristic of molybdoflavoproteins containing iron-sulfur centers. Chlamydomonas XDH was irreversibly inactivated upon incubation of enzyme with its physiological electron donors xanthine and hypoxanthine, in the absence of NAD+, its physiological electron acceptor. As deduced from spectral changes in the 400-500 nm region, xanthine addition provoked enzyme reduction which was followed by inactivation. This irreversible inactivation also took place either under anaerobic conditions or whenever oxygen or any of its derivatives were excluded. Adenine, 8-azaxanthine and acetaldehyde which could act as reducing substrates of XDH were also able to inactivate it upon incubation. The same inactivating effect was observed with NADH and NADPH, electron donors for the diaphorase activity associated with xanthine dehydrogenase. In addition, partial activities of XDH were differently affected by xanthine incubation. We conclude that xanthine dehydrogenase inactivation by substrate is due to an irreversible process affecting mainly molybdenum center and that sequential and uninterrupted electron flow from xanthine to NAD+ is essential to maintain the enzyme in its active form.
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PMID:Purification and substrate inactivation of xanthine dehydrogenase from Chlamydomonas reinhardtii. 152 76

We have tested an ethanol reagent strip developed at the Addiction Research Foundation of Ontario. Alcohol dehydrogenase and nicotinamide adenine dinucleotide, in the presence of pyrazole, react with ethanol to yield acetaldehyde plus reduced nicotinamide adenine dinucleotide. The latter reduces iodonitrotetrazolium chloride in the presence of diaphorase, generating an intense red color. The rate of color development is proportional to the concentration of ethanol. Color is compared at a specific time against a calibrated color scale ranging from green (negative) to red, representing alcohol concentrations of 0, 25, 50, 100, 200, and 400 mg/dl (0-0.4%; 0-87 mmol/liter). We were able to interpolate the color observed between the calibrated blocks. When tested on urine, serum/plasma, and saliva, ethanol concentration determined by the reagent strip correlates well with ethanol concentration as determined by gas chromatography or by automated enzymatic analysis (r = 0.92-0.98, p less than 0.001; slope 0.83-1.16). The reagent strip was shown to be used appropriately by nonexperienced individuals following a 1-min explanation (reagent strip values, r = 0.92; p less than 0.001, slope = 0.97, versus gas chromatography). The reagent strip does not react with methanol (wood alcohol), isopropanol (rubbing alcohol), and ethylene glycol (antifreeze) often found in accidental poisonings. In 379 clinical samples obtained without exclusion criteria from 12 hospital emergency rooms and a liver clinic, the sensitivity of the reagent strip in detecting ethanol was 98%. Specificity was 99%. The reagent strip was found to have virtually unlimited stability under refrigeration (4 degrees C) and to be stable for 3 to 4 months at room temperature (22-23 degrees C).(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Characteristics of a new urine, serum, and saliva alcohol reagent strip. 159 May 43

Incubation of aldehyde dehydrogenase-free mitochondrial preparations with biogenic amines serotonin, tyramine, 2-phenylethylamine and 5-methoxytryptamine resulted in inhibition of enzymes activity of both outer (rotenone-insensitive NADH-cytochrome c reductase) and inner (succinate dehydrogenase, succinate cytochrome c reductase) mitochondrial membranes. Solubilization of mitochondria after the incubation did not influence the amine-induced alteration of succinate dehydrogenase activity. Pretreatment of the organelles with a mixture containing chlorgyline and deprenyl completely inhibited monoamine oxidase (MAO) activity and prevented the effects of all the amines studied on mitochondrial enzymes. MAO-dependent effects of 5-methoxytryptamine were fully reproduced by 5-methoxyindolyl-3-acetaldehyde (one of probable products of 5-methoxytryptamine deamination). The effect of the aldehyde was not prevented by chlorgyline and deprenyl. After selective inhibition of MAO-A by chlorgyline the order of MAO-B-dependent effects of biogenic amines on mitochondrial enzymes studied was as follows: tyramine greater than or equal to 2-phenylethylamine much greater than serotonin. In deprenyl pretreated mitochondria the potency of MAO-A-dependent effects of these amines was: serotonin greater than tyramine much greater than much greater than 2-phenylethylamine. The data obtained suggest that the product(s) of oxidative deamination of biogenic amines (probably the aldehydes) catalyzed by both types of MAO (MAO-A and MAO-B) are able to regulate the energy functions of mitochondria.
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PMID:[The role of monoamine oxidase in the regulation of mitochondrial energy functions]. 175 90

The stereochemical course of the reduction of acetaldehyde to ethanol was investigated by evaluating, with the enzymic system yeast alcohol dehydrogenase/diaphorase and g.c.-m.s., the configuration of [1-2H]ethanol obtained from [1-2H]acetaldehyde with different micro-organisms. Although only S-[1-2H]ethanol was formed, all the micro-organisms showed evidence of the existence of alcohol dehydrogenases with opposite stereospecificity.
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PMID:Reduction of acetaldehyde to ethanol by some micro-organisms and its stereospecificity. 339 Jan 47

The ultrastructure of reduced nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase-positive neurons in cat cerebral cortex, amygdala and caudate nucleus was investigated by electron microscopy using a modified method applicable to aldehyde-fixed tissues. These NADPH diaphorase-positive neurons were morphologically similar to neurons immunohistochemically positive for somatostatin. They had large amounts of electron-dense formazan reaction products scattered through the whole cytoplasm but not in the mitochondria or nucleus. Similar electron-dense reaction products were visible in the dendrites of these neurons. The results indicate that NADPH diaphorase histochemistry is a useful method for the ultrastructural examination of particular groups of neurons.
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PMID:Ultrastructure of reduced nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase-positive neurons in the cat cerebral cortex, amygdala and caudate nucleus. 340 36

Up to now, more than 40.000 determinations of urinary estrogens (E1 + E2) have been carried out in routine clinical analysis by the enzymatic method using estradiol dehydrogenase. This method makes use of the transhydrogenating activity of the placental enzyme: this enzyme transfers hydrogen from NADP to NAD with recycling of the specific substrate (E1 + E2). For several years the necessary reagents have been commercially available in the form of a kit. Nonetheless, various improvements have been made to the measurement of reduced NAD, which accumulates in the reaction medium and is directly proportional to the concentration of the two estrogens. Three protocols are available at present: Spectrophotometric measurement at 340 nm (initial technique); Colorimetric measurement at 492 nm. The pink colour measured arises from the reduction of a tetrazolium salt (INT) by reduced NAD in a coupled system using diaphorase; Measurement by bioluminescence of the light energy liberated on the reduction of flavin derivatives by NADH. The reaction is mediated by various enzymes isolated from marine bacteria (FMN oxidoreductase and luciferase) in the presence of an aliphatic aldehyde (decanal). The procedure for each of these protocols is described as well as the means for controlling the linearity of the reaction. The choice of protocol is determined by the biological fluid available, the speed of response desired and the cost of the analysis.
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PMID:[Various protocols for determining estrogens by the enzymatic method using estradiol dehydrogenase. Respective procedures and advantages]. 386 35

A study was made of the effect of chronic administration of the hypolipidemic drug clofibrate on the activity and intracellular localization of rat liver aldehyde dehydrogenase. The enzyme was assayed using several aliphatic and aromatic aldehydes. Clofibrate treatment caused a 1.5 to 2.3-fold increase in the liver specific aldehyde dehydrogenase activity. The induced enzyme has a high Km for acetaldehyde and was found to be located in peroxisomes and microsomes. Clofibrate did not alter the enzyme activity in the cytoplasmic fraction. The total peroxisomal aldehyde dehydrogenase activity increased 3 to 4-fold under the action of clofibrate. Disruption of the purified peroxisomes by the hypotonic treatment or in the alkaline conditions resulted in the release of catalase from the broken organelles, while aldehyde dehydrogenase as well as nucleoid-bound urate oxidase and the peroxisomal membrane marker NADH:cytochrome c reductase remained in the peroxisomal 'ghosts'. At the same time, treatment by Triton X-100 led to solubilization of the membrane-bound NADH:cytochrome c reductase and aldehyde dehydrogenase from intact peroxisomes and their 'ghosts'. These results indicate that aldehyde dehydrogenase is located in the peroxisomal membrane. The peroxisomal aldehyde dehydrogenase is active with different aliphatic and aromatic aldehydes, except for formaldehyde and glyceraldehyde. The enzyme Km values lie in the millimolar range for acetaldehyde, propionaldehyde, benzaldehyde and phenylacetaldehyde and in the micromolar range for nonanal. Both NAD and NADP serve as coenzymes for the enzyme. Aldehyde dehydrogenase was inhibited by disulfiram, N-ethylmaleimide and 5,5'-dithiobis(2-nitrobenzoic)acid. According to its basic kinetic properties peroxisomal aldehyde dehydrogenase seems to be similar to a clofibrate-induced microsomal enzyme. The functional role of both enzymes in the liver cells is discussed.
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PMID:Intraparticulate localization and some properties of a clofibrate-induced peroxisomal aldehyde dehydrogenase from rat liver. 399 98

Baboons fed ethanol (50% of total calories) chronically develop ultrastructural alterations of hepatic mitochondria. To determine whether mitochondrial functions are also altered, mitochondria were isolated from nine baboons fed ethanol chronically and their pair-fed controls. At the fatty liver stage, ADP-stimulated respiration was depressed in ethanol-fed baboons by 59.4% with glutamate, 43.2% with acetaldehyde, 45.1% with succinate and 51.1% with ascorbate as substrates. A similar decrease was noted in the ADP/O ratio (14 to 28%) and respiratory control ratio (20 to 44%) with all substrates. Similar alterations of mitochondrial functions were observed in baboons with more advanced stages of liver disease, namely fibrosis. These changes after ethanol treatment were associated with decreases in the enzyme activities of mitochondrial respiratory chain: glutamate, NADH and succinate dehydrogenase (42, 24 and 28%, respectively), glutamate-, NADH- or succinate-cytochrome c reductase (42, 27 and 32%, respectively) and cytochrome oxidase (59.6%). The content of all cytochromes was also decreased in ethanol-fed baboons, especially aa3 (57%). Moreover, [14C]leucine incorporation into mitochondrial membranes was depressed by 21% after ethanol treatment. On the other hand, glutamate dehydrogenase activities of serum and cytosol in ethanol-fed baboons were significantly higher than those in pair-fed controls. Morphologically, mitochondria of ethanol-fed baboons were larger than those of pair-fed controls. However, the mitochondrial protein content per mitochondrial DNA was unchanged. From these results, we conclude that, morphologically and functionally, hepatic mitochondria in baboons are altered by chronic ethanol consumption; it is noteworthy that these changes are fully developed already at the fatty liver stage, and that morphological alteration appears to reflect the damage of mitochondrial membranes rather than an adaptive hypertrophy.
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PMID:Biochemical and morphological alterations of baboon hepatic mitochondria after chronic ethanol consumption. 653 46

Wistar male rats were exposed by inhalation to 50, 100 or 400 ppm of ethylene glycol monomethyl ether (EGME) for 1 to 2 weeks. The overall hepatic drug oxidation reactions, O-deethylation of 7-ethoxycoumarin and 7-ethoxyresorufin and cytochrome P-450 content were only slightly affected by the EGME exposures. NADPH cytochrome c reductase activity showed a tendency toward a dose-dependent decrease in liver, the activity being 73% and 64% of that in the controls after one and two weeks of exposure, at 400 ppm respectively. UDP glucuronosyl transferase activity exhibited a dose-dependent enhancement in liver microsomes after exposure for two weeks to EGME. The enhancement was 1.3- 1.7- and 3.0 fold with exposure to 50, 100 and 400 ppm of EGME respectively. After exposure for one week the UDPglucuronosyltransferase activity in kidney microsomes was similarly enhanced. A dose-related increase in measurable UDPglucuronosyltransferase activity was also obtained in Triton X-100 treated hepatic microsomes. GSH levels of the liver and kidneys in EGME treated animals showed a tendency towards a dose-dependent increase. The activities of low-Km and high-Km aldehyde dehydrogenases in liver were decreased 6 - 14% of that in the controls with exposure to 400 ppm of EGME when glycolaldehyde was used as a substrate. Serum alanine aminotransferase activity was not influenced by inhalation exposures to EGME.
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PMID:Dose-dependent toxicity of ethylene glycol monomethyl ether vapour in the rat. 680 Jul 97

The effect of aldehyde fixation on NADPH- and NADH-dependent diaphorase (d) histochemistry and nitric oxide synthase (NOS) immunocytochemistry in the brain was investigated by comparing the distribution of these enzymes in in situ nitrocellulose blots of unfixed brain sections with that in aldehyde-fixed brain sections. Substitution of NADPH by NADH yielded no gross differences in cellular distribution in the native blot, whereas in fixed sections NADH produced nonspecific staining of the entire section. In the in situ blot NADPHd histochemistry therefore visualized general nitroblue tetrazolium reductase (NBTr) activity, which was particularly strong in hippocampal pyramidal neurons and cerebellar Purkinje cells. Aldehyde fixation abolished the anatomical pattern of general NBTr activity and changed the histochemical distribution in that of the NADPHd activity associated with the distribution of NOS-I immunoreactivity (ir). Fixation intensified NADPHd histochem- ical staining in specific neurons, resulting in outstanding, Golgi-like staining of these neurons in several brain regions, whereas the general NBTr activity in pyramidal and Purkinje cells disappeared. In contrast to the histochemical diaphorase distribution, the distribution of NOS-I ir on blots and in aldehyde-fixed brain sections was similar. No NOS was observed in hippocampal pyramidal and cerebellar Purkinje neurons. In regions like cerebral and cerebellar cortex and striatum the applied anti NOS-I serum had a higher affinity for the native protein. It is concluded that aldehydes, rather than to progressively suppress NOS-unrelated enzymes, differentially elicit NADPHd activity in some groups of neurons while leaving NOS-ir unaffected.
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PMID:Aldehyde fixation differentially affects distribution of diaphorase activity but not of nitric oxide synthase immunoreactivity in rat brain. 866 71


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