UMLS:C0027960 - FACTA Search

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Query: UMLS:C0027960 (mole)
21,279 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

An automated AutoAnalyzer method using 5:5'-dithiobis-2-nitrobenzoic acid is described for determining whole blood glutathione reductase (BGR) activity and for measuring in vitro activation of BGR with flavin adenine dinucleotide (FAD). BGR activity is expressed as mumoles glutathione regenerated from oxidized glutathione per ml of whole blood (WB) or per g of hemoglobin. The stimulatory effect of FAD on BGR activity divided by the activity without FAD determined the activity coefficient (AC). We found that NADPH and oxidized glutathione assay concentrations of 0.100 mmole/liter and 0.250 mmole/liter, respectively, in 0.1 mole/liter phosphate buffer, pH 7.4, gave consistent results when WB, before assay, was diluted 20-fold. WB samples to be stored are initially diluted 10-fold with distilled water and frozen. Prior to assay, two aliquots of the sample are diluted 2-fold, one aliquot with distilled water and another with 46 mumole/liter FAD. With sample and manifold dilutions the assay FAD concentrations is 1.0 mumole/liter: assay concentrations greater than 5.0 mumole FAD/liter were shown to be inhibitory. We examined blood samples from 617 children in the age range 6 to 60 months and determined the normal AC range to be between 1.00 and 1.35. Six weaned rats (23 days of age), maintained on a riboflavin-deficient diet, showed a mean AC of 1.23, 1.54, 2.02, and 2.41 at 23, 26, 30, and 36 days of age, respectively. Six control rats maintained an AC of 1.23 +/- 0.05 (SD) during the same period.
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PMID:An automated flavin adenine dinucleotide-dependent glutathione reductase assay for assessing riboflavin nutriture. 0 81

African trypanosomes are devoid of glutathione reductase activity, and instead contain a unique flavoprotein variant, trypanothione reductase, which acts on a cyclic derivative of glutathione, trypanothione. The high degree of sequence similarity between trypanothione reductase and glutathione reductase, as well as the obvious similarity in the reactions catalyzed, led us to investigate the pH dependence of the kinetic parameters, and the isotopic behavior of trypanothione reductase. The pH dependence of the kinetic parameters V, V/K for NADH, and V/K for oxidized trypanothione has been determined for trypanothione reductase from Trypanosoma congolense. Both V/K for NADH and the maximum velocity decrease as single groups exhibiting pK values of 8.87 +/- 0.09 and 9.45 +/- 0.07, respectively, are deprotonated. V/K for oxidized trypanothione, T(S)2, decreases as two groups exhibiting experimentally indistinguishable pK values of 8.74 +/- 0.03 are deprotonated. Variable magnitudes of the primary deuterium kinetic isotope effects on pyridine nucleotide oxidation are observed on V and V/K when different pyridine nucleotide substrates are used, and the magnitude of DV and D(V/K) is independent of the oxidized trypanothione concentration at pH 7.25. Solvent kinetic isotope effects, obtained with 2',3'-cNADPH as the variable substrate, were observed on V only, and plots of V versus mole fraction of D2O (i.e., proton inventory) were linear, and yielded values of 1.3-1.6 for D2OV. Solvent kinetic isotope effects obtained with alternate pyridine nucleotides as substrates were also observed on V, and the magnitude of D2OV decreases for each pyridine nucleotide as its maximal velocity relative to that of NADPH oxidation decreases.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Kinetic isotope effect analysis of the reaction catalyzed by Trypanosoma congolense trypanothione reductase. 163 54

Glutathione reductase catalyzes the NADPH-dependent reduction of oxidized glutathione (GSSG). The kinetic mechanism is ping-pong, and we have investigated the rate-limiting nature of proton-transfer steps in the reactions catalyzed by the spinach, yeast, and human erythrocyte glutathione reductases using a combination of alternate substrate and solvent kinetic isotope effects. With NADPH or GSSG as the variable substrate, at a fixed, saturating concentration of the other substrate, solvent kinetic isotope effects were observed on V but not V/K. Plots of Vm vs mole fraction of D2O (proton inventories) were linear in both cases for the yeast, spinach, and human erythrocyte enzymes. When solvent kinetic isotope effect studies were performed with DTNB instead of GSSG as an alternate substrate, a solvent kinetic isotope effect of 1.0 was observed. Solvent kinetic isotope effect measurements were also performed on the asymmetric disulfides GSSNB and GSSNP by using human erythrocyte glutathione reductase. The Km values for GSSNB and GSSNP were 70 microM and 13 microM, respectively, and V values were 62 and 57% of the one calculated for GSSG, respectively. Both of these substrates yield solvent kinetic isotope effects greater than 1.0 on both V and V/K and linear proton inventories, indicating that a single proton-transfer step is still rate limiting. These data are discussed in relationship to the chemical mechanism of GSSG reduction and the identity of the proton-transfer step whose rate is sensitive to solvent isotopic composition. Finally, the solvent equilibrium isotope effect measured with yeast glutathione reductase is 4.98, which allows us to calculate a fractionation factor for the thiol moiety of GSH of 0.456.
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PMID:Glutathione reductase: solvent equilibrium and kinetic isotope effects. 284 77

Pure glutathione reductase from Saccharomyces cerevisiae catalyzed under anaerobic conditions the enzymatic reduction of GSSG using electrochemically reduced methyl viologen as electron donor. The new assay was completely dependent on the amount of active enzyme present, and involved the formation of 1 mol GSH per mole of reduced methyl viologen consumed. The enzyme followed a standard Michaelis-Menten kinetics; a Km = 230 microM for reduced methyl viologen and a turnover number of 969 mumol GSSG reduced per minute per micromole enzyme were determined. The enzymatic activity seemed to depend on the redox potential, showing half-maximal activity at -0.407 V. The enzyme was quite specific: the activity using reduced benzyl viologen as electron donor was just 1.5% of that obtained with reduced methyl viologen at the same concentration and potential. Glutathione reductase was totally inactivated after a brief anaerobic exposure with reduced methyl viologen in the absence of GSSG; a partial reactivation was observed following addition of glutathione disulfide. No inhibition of the methyl viologen-dependent activity was observed in the presence of 2',5'-ADP or 2'-P-5'-ADP-ribose, two NADP(H) analogs, at concentrations which drastically inhibited the NADPH-dependent activity, thus suggesting that the reduced viologen does not interact with the pyridine nucleotide-binding site.
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PMID:Electron transfer between reduced methyl viologen and oxidized glutathione: a new assay of Saccharomyces cerevisiae glutathione reductase. 353 78

Cystine reduction in Streptococcus agalactiae, resulting in sulfhydryl formation, may account for antagonism of the antibacterial effect of lactoperoxidase-thiocyanate-hydrogen peroxide when cystine is present in excess of the amount needed for maximum growth. Accumulation of cystine by S. agalactiae and its reduction to form sulfhydryl compounds were demonstrated. The reduction of cystine appeared to occur by a couple reaction between glutathione reductase and glutathione-disulfide transhydrogenase activity, both of which were found in the supernatant fraction from cell homogenates. NADPH-specific glutathione reductase activity was found in the pellet and supernatant fractions from cell homogenates. Two sulfhydryls were formed for each mole of NADPH used during cystine reduction. The information presented offers a plausible explanation of how cystine, when present in excess of growth needs, may be reduced to generate sulfhydryl compounds which neutralize the antibacterial effect of lactoperoxidase-thiocyanate-hydrogen peroxide on S. agalactiae.
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PMID:Cystine antagonism of the antibacterial action of lactoperoxidase-thiocyanate-hydrogen peroxide on Streptococcus agalactiae. 637 Jan 35

Reactive coenzyme analogues omega-(3-diazoniumpyridinium)alkyl adenosine diphosphate were prepared by reaction of omega-(3-aminopyridinium)alkyl adenosine diphosphate with nitrous acid. In these compounds the nicotinamide ribose is substituted by hydrocarbon chains of varied lengths (n-ethyl to n-pentyl). The diazonium compounds are very unstable and decompose rapidly at room temperature. They show a better stability to 0 degree C. Lactate and alcohol dehydrogenase do not react with any of the analogues. Glyceraldehyde-3-phosphate dehydrogenase reacts rapidly with the diazoniumpentyl compound. Decreasing the length of the alkyl chain significantly decreases the inactivation velocity. 3 alpha, 20 beta-Hydroxysteroid dehydrogenase reacts at 0 degree C with the ethyl homologue and slowly with the propyl compound. The butyl- and pentyl analogues do not inactivate at 0 degree C. Tests with 14C-labeled 2-(3-diazoniumpyridinium)ethyl adenosine diphosphate show that complete loss of enzyme activity results after incorporation of 2 moles of inactivator into 1 mole of tetrameric enzyme. 4-(3-Acetylpyridinium)butyl 2'-phospho-adenosine diphosphate, a structural analogue of NADP+, was prepared by condensation of adenosine-2,3-cyclophospho-5'-phosphomorpholidate with (3-acetylpyridinium)butyl phosphate, followed by hydrolysis of the cyclic phosphoric acid with 2':3'-cyclonucleotide-3'-phosphodiesterase. Because of the redox potential (-315 mV) and the distance between the pyridinium and phosphate groups, this analogue is a hydrogen acceptor and its reduced form a hydrogen donor in tests with alcohol dehydrogenase from Thermoanaerobium brockii. The reduced form of the coenzyme analogue also is a hydrogen donor with glutathione reductase. With other NADP+-dependent dehydrogenases the compound has been shown to be a competitive inhibitor against the natural coenzyme. The acetyl group reacts with bromine to form the bromoacetyl group. This reactive bromoacetyl analogue is a specific active-site directed irreversible inhibitor of isocitrate dehydrogenase.
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PMID:New reactive coenzyme analogues for affinity labeling of NAD+ and NADP+ dependent dehydrogenases. 754 38

Yeast glutathione reductase is inactivated by pyridoxal 5'-phosphate (PLP). The reactivation of the enzyme by dilution as well as a characteristic absorption peak at 325 nm exhibited by NaBH4-reduced-PLP modified enzyme show that the inactivation is due to the specific modification of the epsilon-amino group of lysine residue. The maximum of 70% inactivation was observed at 7mM PLP and the equilibrium was reached within 3 min. Kinetic and equilibrium analysis of inactivation data derived at different PLP concentrations showed that a noncovalent intermediate is formed prior to inactivation. From the studies on the effect of pH on the inactivation rate, the pKa of epsilon-amino group of the reactive lysine residue was calculated to be 7.3. Among various protecting agents tried, only NADP was found to be effective. The apparent stoichiometry of the reaction was one to one as the incorporation of 0.65 mole PLP/mole of enzyme led to 70% inactivation at saturating PLP concentration.
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PMID:Modification of an essential amino group of glutathione reductase from yeast by pyridoxal 5'-phosphate. 924 56

The mechanism of oxidation or reduction using the electron method was investigated for (I) aniline; (II) nitrobenzene; (III) nitrate; (IV) sulphanilamide; (V) hydrogen peroxide; (VI) hydroxyl free radical; (VII) ferricyanide; (VIII) acetylphenylhydrazine; (IX) nitrite; (X) chlorate and (XI) hydroxylamine respectively. Substances (II), (III), (V), (VI), (VII), (IX), (X) and (XI) evolved as oxidants, with (II), nitrobenzene and (X), chlorate as the most powerful oxidants (number of moles of HbFe(2+)(haemoglobin) of 6 reacting with 1.0 mole of the substance). Substances (I), (IV) and (VII) evolved as reductants of equal reducing power (number of moles of HbFe(3+)(methaemoglobin) of 4 reacting with 1.0 mole of the substance). Using the following equations, the impact of oxidants and reductants on glutathione (GSH) peroxidase, glutathione (GSSC) reductase and NADHmetHb reductase respectively on methaemoglobinaemia generation was investigated. [Equation in text]. Redox potential change (DeltaE' (o)) of 1.77, -1.77 and 1.86 volt and free energy change (DeltaG(o)') of -81, 81 and -85.8 kcal/mol were calculated for GSH peroxidase, GSSG reductase and NADHmetHb reductase systems respectively. In sustained methaemoglobinaemia, these mechanisms predict low levels of NADHmetHb reductase and glutathione peroxidase respectively, but high levels of glutathione reductase in red blood cells on exposure to oxidants. The significance of these mechanisms was investigated in cord blood, neonatal, adult red blood cells and other biological systems. It was concluded that any reaction with a positive DeltaE(o)' and negative DeltaG(o)' with the Fe(3+): Fe(2+)couple will indicate methaemoglobin oxidizing power. The effects on red blood cells and white blood cells were manifested in the biochemical toxicology of nitroso (PhN = 0), arylamine glucuronide (PhNHG) and arene imine respectively.
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PMID:Theoretical mechanistic basis of oxidants of methaemoglobin formation. 1079 Jul 68

The blind subterranean mole rat Spalax ehrenbergi superspecies has evolved adaptive strategies to cope with underground stress. Hypoxia is known to stimulate reactive oxygen species generation; however, mechanisms by which Spalax counteracts oxidative damage have not been investigated before. We studied in Spalax the oxidative status of the Harderian gland (HG), an organ which is particularly vulnerable to oxidative stress in many rodents. With regard to the sexual dimorphism found in this gland, differences between males and females were determined and compared to the surface-dwelling Syrian hamster. Our results show, for the first time, that Spalax exhibits remarkably low biomolecular damage, which implies the existence of physiological strategies to avoid oxidative damage under fluctuating O(2) and CO(2) levels existing in the mole rat's subterranean niche. Correspondingly, main antioxidant enzymes, such as superoxide dismutase (SOD), catalase, and glutathione reductase (GR), exhibited high activities in both genders; in particular, remarkably high levels were measured in SOD. SOD and GR activities showed statistically significant differences between sexes. Melatonin, an important circadian agent is also a very important antioxidant molecule and is synthesized in the Harderian glands (HGs) of Spalax. Therefore, the possible interaction between antioxidant enzymes and melatonin is suggested.
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PMID:Antioxidant activity in Spalax ehrenbergi: a possible adaptation to underground stress. 1647 5

Ascorbic acid oxidase activity in Myrothecium verrucaria extracts resulted in O(2) uptake exceeding 0.5 mole per mole of ascorbic acid and in CO(2) evolution. Measurement of oxidized ascorbic acid at completion of the reaction demonstrated that an average of 10% of the oxidized product disappeared. A comparison of the gas exchange data with the amount of ascorbic acid not accounted for indicated that the reaction could not be explained by independent oxidase and oxygenase systems. Chromatographic examination of the reaction mixtures identified l-threonic acid. Experiments with ascorbic acid-1-(14)C showed that C-1 was partially decarboxylated during the oxidation. Test of the fungal extracts for enzymes that might explain the deviation from expected stoichiometry showed that phenolase, glutathione reductase, cytochrome oxidase, peroxidase and oxalic decarboxylase were not involved. Addition of azide in concentrations sufficient to block catalase increased excess O(2) consumption about 65%. No enzymes were found that could directly attack oxidized ascorbic acid. H(2)O(2) accumulated during oxidation in azide-blocked systems.The O(2) excess could be explained by assuming the enzyme had peroxidative capacity on a reductant other than ascorbic acid. An intermediate of ascorbic acid oxidation appeared to function as the substrate yielding CO(2) and l-threonic acid on degradation. The increase in excess O(2) utilized in azide-blocked systems and the H(2)O(2) accumulation also were explained by the proposed scheme.Another interpretation would involve production of free radicals during ascorbic acid oxidation. Evidence for this was the ability of extracts to oxidize DPNH in the presence of ascorbic acid. Oxygen radicals formed in such reactions were considered possible agents of degradation of ascorbic acid.
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PMID:Reaction Properties of the Ascorbic Acid Oxidase from Myrothecium verrucaria. 1665 40

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