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:1.6.5.2 (NQO1)
6,196 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

1. Dihydrodiol dehydrogenase activities were investigated in rabbit liver. Using a five-step purification scheme, eight isoenzymes of dihydrodiol dehydrogenase with isoelectric points of 5.55-9.3 and promoter molecular masses of 34-35 kDa were purified to apparent homogeneity and designated CF-1 to CF-6, CM-1 and CM-2. 2. CF-1 and CF-2 had near-neutral isoelectric points of 7.4 and 6.8 and molecular masses of about 125 kDa in the native state. Both enzymes readily accepted NAD+ as well as NADP+ as coenzymes, had relatively low Km values of 0.33 mM and 0.47 mM for benzene dihydrodiol and resembled previously described carbonyl reductases in their substrate specificity towards ketones and quinones. 3. CF-5 and CF-6 had acidic isoelectric points of 5.9 and 5.55 and native molecular masses of approximately 60 kDa. They displayed a strong preference for NADP(H) as coenzyme and had high Km and Vmax with benzene dihydrodiol. Since these enzymes reduced p-nitrobenzaldehyde and glucuronic acid efficiently, they appeared to be closely related to aldehyde reductase. 4. CF-4 had a high 3 alpha-hydroxysteroid dehydrogenase activity for the diagnostic substrate androsterone, a moderate activity for other 3 alpha-hydroxysteroids as well as 17 alpha-hydroxysteroids, and relatively low activities for 3 beta-hydroxysteroids and 17 beta-hydroxysteroids. CF-5 and CM-1 had high 17 beta-hydroxysteroid dehydrogenase activity for the diagnostic substrate 5 alpha-dihydrotestosterone, and low to moderate activities for other 17 beta-hydroxysteroids as well as 3 alpha-hydroxysteroids. 5. The isoenzyme CM-2 had an isoelectric point of 9.3 and was a very active quinone reductase with phenanthrene-9,10-quinone as substrate. It was potently inhibited by phenobarbital. 6. We conclude that the dihydrodiol dehydrogenase activities of rabbit liver are associated with aldehyde and carbonyl reductase and with 3 alpha-hydroxysteroid and 17 beta-hydroxysteroid dehydrogenases.
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PMID:Dihydrodiol dehydrogenase activities of rabbit liver are associated with hydroxysteroid dehydrogenases and aldo-keto reductases. 157 98

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

The increasing concern and the efforts in determining neurological effects in offsprings resulting from maternal exposure to xenobiotics are faced with several difficulties in monitoring damage to the central nervous system. In this paper, the efficiency of several enzyme histochemical reactions for analysing the forebrain and the trigeminal ganglia of rat foetuses are reported. Brains of 20-day-old Sprague-Dawley rat foetuses were frozen and analysed for 18 enzymes that had previously been used to monitor initial injury caused by toxic compounds in liver and other organs. Eight enzymes appeared suitable as histochemical markers for the functional integrity of different areas in brain and ganglia of rats exposed to xenobiotics. They were lactate, malate, glycerophosphate (NAD-linked), succinate, aldehyde and glucose 6-phosphate dehydrogenases, alpha-glycerophosphate-menadione oxidoreductase and cytochrome c oxidase. The activities of the enzymes were determined by microphotometry and the arrangement of absorbances of the enzyme final reaction products into appropriate analytical tables is proposed as an efficient procedure for data analysis.
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PMID:Quantitative enzyme histochemistry of rat foetal brain and trigeminal ganglion. 297 23

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

Liver cytosolic fractions are known to catalyze the reduction of certain C-nitroso compounds to their corresponding hydroxylamines and amines. Alcohol dehydrogenase (ADH), NAD(P)H:quinone oxidoreductase, and xanthine and aldehyde oxidases have been implicated as C-nitroso reductases. To probe the role of these cytosolic enzymes in the reduction of C-nitroso compounds we have studied the effects of classical inhibitors of these enzymes on the ability of liver cytosolic fractions from ADH+ and ADH- deermice to reduce p-nitrosophenol to p-aminophenol. Pyrazole, a potent inhibitor of ADH, inhibited NADH-p-nitrosophenol reduction by ADH+ cytosol by > 85%. Thus, ADH contributes substantially to NADH-C-nitroso reduction by cytosol from ADH+ deermice. The NAD(P)H:quinone oxidoreductase inhibitor, dicumarol, inhibited NADH-dependent p-aminophenol formation by about 25%; however, dicumarol potently inhibited the NADPH-dependent formation (90-95%). As expected, cytosol from ADH- deermice did not catalyze pyrazole-sensitive (ADH-dependent) C-nitroso reduction with NADH as the cofactor. Both NADPH- and NADH-p-nitrosophenol reduction by ADH- cytosol were inhibited > 90% by dicumarol. The xanthine oxidase/aldehyde oxidase inhibitor, allopurinol, was without effect on NAD(P)H cytosolic p-nitrosophenol reduction from ADH- and ADH+ deermice under either aerobic or anaerobic conditions. Our findings suggest that in the ADH+ animal, ADH contributes significantly to NADH-dependent C-nitroso reduction by cytosol relative to NAD(P)H:quinone oxidoreductase. NADPH-dependent p-nitrosophenol reduction by liver cytosol of ADH+ animals is mostly dicumarol-sensitive, which implicates NAD(P)H:quinone oxidoreductase as the major NADPH-dependent activity. In ADH- deermice, both NADH- and NADPH-dependent p-nitrosophenol reduction are essentially dicumarol-sensitive (NAD(P)H:quinone oxidoreductase-dependent). Because the toxic expression of C-nitroso compounds is mediated by hydroxylamine intermediates, the present data indicate the importance of considering the role of ADH in the toxic sequelae of nitro and nitroso arenes.
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PMID:p-nitrosophenol reduction by liver cytosol from ADH-positive and -negative deermice (Peromyscus maniculatus). 753 87

The mechanism of reduction of p-nitrosophenol (pNSP) catalyzed by horse liver alcohol dehydrogenase (HADH) and human pi-alcohol dehydrogenase (pi-ADH) has been compared in transient and steady-state experiments. Our results indicate that pNSP reduction catalyzed by these two ADH proceeds by different mechanisms. In one mechanism, shown by Equation 1, pNSP is reduced to p-aminophenol (pAP) via two enzymatic steps (Steps 1 and 3), which are mediated by the nonenzymatic dehydration of p-N-hydroxyaminophenol (pN-OHAP) to 1,4-benzoquinoneimine (BQI) (Step 2). [formula: see text] Pathway (I) is proposed mainly for pi-ADH but can be catalyzed by HADH. However, Step 3 is catalyzed approximately 2 orders of magnitude more slowly by HADH than by pi-ADH. This conclusion is confirmed by the results, which indicate that pi-ADH very efficiently catalyzes the reduction of BQI and 1,4-benzoquinone (BQ) to the corresponding hydroquinones. The kinetic constants determined at pH 7.4 suggest that pi-ADH is a more efficient quinone reductase and nitroso reductase than it is an ethanol oxidase or acetaldehyde reductase. An alternative mechanism of pNSP reduction, shown by Equation 2, is suggested for HADH. In this mechanism, formation of the p-hydroxybenzylnitrenium ion (pNH+P) occurs at the active-site zinc ion of the enzyme (Step 2) and accelerates further nonenzymatic reduction to pAP or hydrolysis to BQ (Step 3). [formula: see text]
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PMID:Mechanism of p-nitrosophenol reduction catalyzed by horse liver and human pi-alcohol dehydrogenase (ADH). Human pi-ADH as a quinone reductase. 798 27

The class-3 aldehyde dehydrogenase that is overexpressed (> 100-fold) in human breast adenocarcinoma MCF-7/0 cells made resistant (> 30-fold as judged by LC90s) to oxazaphosphorines, such as mafosfamide, by growing them in the presence of polycyclic aromatic hydrocarbons, e.g., methylcholanthrene (3 microM for 5 days), was isolated and characterized. Its physical and catalytic properties were identical to those of the prototypical human stomach mucosa cytosolic class-3 aldehyde dehydrogenase, type-1 ALDH-3, except that it catalyzed, though not very rapidly, the oxidation of aldophosphamide, whereas the stomach mucosa enzyme essentially did not; hence, it was judged to be a slight variant of the prototypical enzyme. Carcinogens that are not ligands for the Ah receptor, barbiturates known to induce hepatic cytochrome P450s, steroid hormones, an antiestrogen, and oxazaphosphorines did not induce the enzyme or the largely oxazaphosphorine-specific acquired resistance. Whereas methylcholanthrene induced (a) resistance to mafosfamide and (b) class-3 aldehyde dehydrogenase activity, as well as glutathione S-transferase and DT-diaphorase activities, in the estrogen receptor-positive MCF-7/0 cells, it did not do so in two other human breast adenocarcinoma cell lines, MDA-MB-231 and SK-BR-3, each of which is estrogen receptor negative. Expression of the class-3 aldehyde dehydrogenase and the loss of sensitivity to mafosfamide by polycyclic aromatic hydrocarbon-treated MCF-7/0 cells were transient; each returned to essentially basal levels within 15 days when the polycyclic aromatic hydrocarbon was removed from the culture medium. Insensitivity to the oxazaphosphorines on the part of polycyclic aromatic hydrocarbon-treated MCF-7/0 cells was not observed when exposure to mafosfamide (30 min) was in the presence of benzaldehyde or octanal, each a relatively good substrate for cytosolic class-3 aldehyde dehydrogenases, whereas it was retained when exposure to mafosfamide was in the presence of acetaldehyde, a relatively poor substrate for these enzymes. These observations demonstrate that ligands for the Ah receptor can induce a transient, largely oxazaphosphorine-specific, acquired cellular resistance, and they are consistent with the notion that elevated levels of a cytosolic class-3 aldehyde dehydrogenase nearly identical to the prototypical type-1 class-3 aldehyde dehydrogenase expressed by human stomach mucosa account for the Ah receptor ligand-induced oxazaphosphorine-specific acquired resistance, most probably by catalyzing the detoxification of aldophosphamide.
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PMID:Identification of a methylcholanthrene-induced aldehyde dehydrogenase in a human breast adenocarcinoma cell line exhibiting oxazaphosphorine-specific acquired resistance. 817 25


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