A modified method of estimation of xanthine oxidase in tissue homogenates.
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Coloring Agents↗
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Kinetic enzymic method for automated determination of total cholesterol in serum.
We describe a rapid, kinetic, fixed-time method for determining serum total cholesterol by use of cholesterol esterase, cholesterol oxidase, and the indicator reaction with peroxidase, 4-aminophenazone, and phenol. On addition of the competitive inhibitor 3,4-dichlorophenol the Michaelis constant of cholesterol oxidase is apparently increased, which extends the linear relation between absorbance change and cholesterol concentration to 20.7 to 25.9 mmol/L, depending on the analyzer being used. For calibration, a single standard is used. Total analysis time is in the range of 80 to 210 s. Incubation temperature is 25 degrees C or 37 degrees C. The single-reagent procedure has been adapted to three different centrifugal analyzers and to the Eppendorf ACP 5040 analyzer. It yields precise and accurate results and is insensitive to potential interferences.
3-Hydroxysteroid Dehydrogenases↗
Restoration of antioxidants in liver by methionine feeding in experimental rat urolithiasis.
The effect of methionine or citrate on antioxidant defense system has been studied in urolithic rat. Liver weight and its protein concentration did not change in the rats fed with calculi producing diet (CPD) when compared to normal diet fed rats. Feeding rats along with citrate (c-CPD) or methionine (m-CPD) improved their body weight gain. Liver microsomes and mitochondria fractions of CPD and c-CPD fed groups showed increased susceptibility for lipid peroxidation in presence of ascorbate and t-butyl hydroperoxide when compared to either control or m-CPD fed groups. Increased superoxide dismutase and xanthine oxidase activities, decreased catalase, glutathione peroxidase and glucose-6-phosphate dehydrogenase activities, decreased concentrations of reduced glutathione, total thiols, ascorbic acid and vitamin-E and increased formation of hydroxyl radical, hydroperoxides and diene conjugates were observed in the liver of both CPD fed group as well as c-CPD fed group. Except SOD and xanthine oxidase, all other parameters were normalized in m-CPD fed group. This suggested that feeding methionine reduced the susceptibility for lipid peroxidation by restoration of the level of free radical scavengers.
Animals↗
Bactericidal agents generated by the peroxidase-catalyzed oxidation of para-hydroquinones.
For the three Gram-negative bacteria, Pseudomonas fluorescens, Escherichia coli, and Erwinia amylovora, p-benzoquinone was the principal bactericidal agent formed in vitro during the oxidation of hydroquinone by horseradish peroxidase, whereas no toxicity could be associated with either phenolic or oxygen-free radicals. Even the continuous generation of p-benzosemiquinone during the simultaneous reduction of p-benzoquinone by xanthine oxidase and reoxidation of hydroquinone by peroxidase was no more toxic than p-benzoquinone alone. Anaerobiosis had no effect on the toxicity of either p-benzoquinone or the peroxidase reaction and the generation of superoxide and hydroxyl radicals catalyzed by xanthine oxidase was not bactericidal. Substitutions on the p-benzoquinone ring decreased quinone toxicity in rough proportion to the decrease in quinone redox potential, suggesting that strong oxidizing potentials are important for such quinone toxicity.
Aerobiosis↗
The reaction of oxygen with radicals from oxidation of tryptophan and indole-3-acetic acid.
The oxidation of tryptophan and indole-3-acetic acid (IAA) by the dibromine radical anion or peroxidase from horseradish in aqueous solution was investigated and compared, especially with respect to the involvement of oxygen and superoxide. Using EPR with spin-trapping, the tryptophanyl radical, generated by either method was found to react with oxygen, although this reaction is too slow to be observed by pulse radiolysis (k < 5 x 10(6) dm3 mol-1 s-1). No superoxide results from this reaction, thus excluding an electron-transfer mechanism and suggesting the formation of a tryptophan peroxyl radical, possibly in a reversible process. These observations imply that in proteins where the tryptophanyl radical exists as a stable species it must either have its reactivity modified by the protein environment or be inaccessible to oxygen. The related molecule LAA is oxidized by either peroxidase or Br2.- to a radical cation that decarboxylates to yield a skatolyl radical. The latter reacts with oxygen to give a peroxyl radical that does not release superoxide. However, O2.- is formed during the peroxidase-catalyzed oxidation of indoleacetic acid. This supports the hypothesis that the peroxidase can act in an oxidase cycle involving ferrous enzyme and compound III, with superoxide as a product.
Anions↗
Oxygen Consumption by Desulfovibrio Strains with and without Polyglucose.
The kinetics of oxygen reduction by Desulfovibrio salexigens Mast1 and the role of polyglucose in this activity were examined and compared with those of strains of D. desulfuricans and D. gigas. Oxidation rates were highest at air saturation (up to 40 nmol of O(2) min mg of protein) and declined with decreasing oxygen concentrations. Studies with cell extracts (CE) indicated that NADH oxidase was entirely responsible for the oxygen reduction in strain Mast1. In D. desulfuricans CSN, at least three independent systems appeared to reduce oxygen. Two were active at all oxygen concentrations (NADH oxidase and NADPH oxidase), and one was maximally active at less than 10 muM oxygen. In contrast to D. gigas and D. salexigens strains, the D. desulfuricans strains also contained NADH peroxidase and NADPH peroxidase activities and did not accumulate polyglucose under nonlimiting growth conditions. At air saturation, initial activities of the oxidases and peroxidases of cells harvested at the end of the log phase were on the order of 20 to 140 nmol of O(2) min mg of protein. In all strains, these enzymes were relatively stable but were susceptible to inactivation as soon as substrates were added to the assay mixture. Under those conditions, all oxidation activity disappeared after ca. 1 h of incubation. The same finding was observed with whole cells of D. desulfuricans CSN and D. desulfuricans ATCC 27774, but inactivation was less pronounced with cells of D. salexigens Mast1. It appeared that the presence of polyglucose in the whole cells retarded the process of inactivation of NADH oxidase, but this property was lost in crude CE. In spite of the effect of polyglucose on the oxidative potential, oxygen-dependent growth of D. salexigens Mast1 could be demonstrated neither in batch nor in continuous culture.
Journal Article↗
The sarcosine oxidase in adapted and unadapted cultures of a strain of Pseudomonas aeruginosa.
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Oxidoreductases↗
Purification and characterization of an NADH oxidase from extremely thermophilic anaerobic bacterium Thermotoga hypogea.
Thermotoga hypogea is an extremely thermophilic anaerobic bacterium capable of growing at 90 degrees C. It was found to be able to grow in the presence of micromolar molecular oxygen (O2). Activity of NADH oxidase was detected in the cell-free extract of T. hypogea, from which an NADH oxidase was purified to homogeneity. The purified enzyme was a homodimeric flavoprotein with a subunit of 50 kDa, revealed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It catalyzed the reduction of O2 to hydrogen peroxide (H2O2), specifically using NADH as electron donor. Its catalytic properties showed that the NADH oxidase had an apparent Vmax value of 37 micromol NADH oxidized min(-1) mg(-1) protein. Apparent Km values for NADH and O2 were determined to be 7.5 microM and 85 microM, respectively. The enzyme exhibited a pH optimum of 7.0 and temperature optimum above 85 degrees C. The NADH-dependent peroxidase activity was also present in the cell-free extract, which could reduce H2O2 produced by the NADH oxidase to H2O. It seems possible that O2 can be reduced to H2O by the oxidase and peroxidase, but further investigation is required to conclude firmly if the purified NADH oxidase is part of an enzyme system that protects anaerobic T. hypogea from accidental exposure to O2.
Flavin-Adenine Dinucleotide↗
[Oxidase and whooping-cough; its study in treatment by high altitude flying].
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Altitude↗
A new method using p-benzoquinone for coupling antigens and antibodies to marker substances.
A method using p-benzoquinone for coupling antigens and antibodies to enzymes and erythrocytes is described. The method involves the treatment of proteins (or polysaccharides) at pH 6 or 7 with an excess of p-benzoquinone. After removal of the unreacted reagent by gel filtration, the "activated" proteins were coupled at pH 8-9 with enzymes or erythrocytes. Biological activities of the proteins were not substantially modified by this treatment since 80-100% of the antigen binding capacity was found to be preserved in p-benzoquinone treated antibodies or Fab fragments. Anti-Ig antibodies (or Fab) were coupled by this procedure to peroxidase, alkaline phosphatase, lactoperoxidase, glucose oxidase and beta-galactosidase, and the conjugates obtained were found to be highly effective in detecting intracellular Ig by immunohistochemical techniques. Erythrocytes coated with sheep anti-mouse Ig antibody or Fab were used to titrate by passive hemagglutination serum Ig. The same erythrocytes were employed to detect by plaque assay mouse Ig secreting cells. Erythrocytes coated with peroxidase, alkaline phosphatase, bovine serum albumin, ribonuclease, Salmonella polysaccharide (B 27 +) and pneumoccocal polysaccharide SIII were employed to titrate serum antibody by passive hemagglutination and hemolysis and to detect mouse antibody secreting cells by plaque assay. All the antigens and antibodies coated erythrocytes prepared gave highly satisfactory and reproducible results.
Alkaline Phosphatase↗
Expression of nicotinamide adenine dinucleotide phosphate oxidase flavoprotein DUOX genes and proteins in human papillary and follicular thyroid carcinomas.
Duox2, and probably Duox1 are glycoflavoproteins involved in the thyroid H2O2 generator functionally associated to thyroperoxidase (TPO). We investigated both DUOX1 and DUOX2 gene expressions using quantitative reverse transcription-polymerase chain reaction (RT-PCR) in 47 thyroid carcinomas, including 10 paired normal/tumoral tissues. In carcinomas, variations of DUOX1 and DUOX2 mRNA levels were parallel, indicating that control mechanisms of both gene expressions operate in tumors as well as in normal thyroid tissues; DUOX1 expression was in the normal range in 20, was decreased up to 50-fold in 8, and increased up to 7-fold in 19 samples. DUOX2 expression was in the normal range in 15, was decreased up to 200-fold in 10, and increased up to 5-fold in 22 samples. In the 10 paired samples, variations of DUOX and TPO gene expressions were not correlated. We analyzed Duoxl/2 protein expression in 86 tumor samples using an antipeptide antiserum reacting with both Duox proteins. In normal tissue, Duox proteins are localized at the apical pole of thyrocytes, with 40% to 60% of thyrocytes being stained. In the 86 cancer tissues, immunostaining was absent in 19 samples, was low in 32, and normal or even slightly increased in the other 35 samples. The expression of Duox proteins was related to tumor differentiation, being more frequently found in neoplastic tissues that were able to pick up radioiodine, and in those with a detectable expression of sodium iodide symporter (NIS), pendrin and TPO.
Adolescent↗
Does captopril change oxidative stress in puromycin aminonucleoside nephropathy?
Puromycin aminonucleoside (PAN) nephropathy in rats has been induced by the intraperitoneal injections of PAN. One group of animals which received PAN has been treated simultaneously with captopril (angiotensine converting enzyme-ACE-inhibitor) with the aim to test whether continuing treatment with captopril along with PAN injections would be able to modulate the toxic effects of PAN. The third group of rats was given only captopril. Morphological changes in the kidney were evaluated by scanning electron microscopy that showed the loss of podocyte foot processes in the kidney of PAN treated animals but also in the kidney of captopril treated ones as well as in the animals treated with both drugs simultaneously. Reduced glutathione content, catalase, superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), xanthine oxidase activities as well as lipid peroxides were investigated in rat blood and kidney. Captopril given alone produced a significant decrease of plasma lipid peroxides, but it did not show any significant effect on investigated antioxidative factor levels neither in blood nor in the kidney. PAN given alone produced a significant depletion of plasma lipid peroxides, kidney catalase and erythrocyte GSH-Px activity as well as a significant increase of plasma catalase and erythrocyte SOD activity. Treatment of animals with both drugs simultaneously resulted in a significant increase of erythrocyte SOD activity and a significant decrease of plasma lipid peroxides, erythrocyte GSH-Px and kidney SOD activities. Kidney xanthine oxidase activity showed a significant increase in both PAN and PAN plus captopril treated animals in comparison with the values of captopril treated rats. These data suggest that PAN changes the antioxidative factor pattern in rat blood and kidney. Contrary to our expectations that captopril may protect the toxic effects of PAN it only to a certain extent modifies these effects showing protective effect only on tissue catalase activity.
Angiotensin-Converting Enzyme Inhibitors↗
Enzymatic enhancement of the catalytic rate of sulfhydryl oxidase.
The rate of oxidation of glutathione by solubilized sulfhydryl oxidase was significantly enhanced in the presence of horseradish peroxidase (donor:hydrogen-peroxide oxidoreductase, EC 1.11.1.7). This enhancement was proportional to the amount of active peroxidase in the assay, but could not be attributed solely to the oxidation of glutathione catalyzed by the peroxidase. A change in the Soret region of the horseradish peroxidase spectrum was observed when both glutathione and peroxidase were present. Moreover, addition of glutathione to a sulfhydryl oxidase/horseradish peroxidase mixture resulted in a rapid shift of the absorbance maximum from 403 nm to 417 nm. This shift indicates the oxidation of horseradish peroxidase. Spectra for three isozyme preparations of horseradish peroxidase, two acidic and one basic, all underwent this red-shift in the presence of sulfhydryl oxidase and glutathione. Cysteine and N-acetylcysteine could replace glutathione. Addition of catalase had no effect on the oxidation of peroxidase, indicating that the peroxide involved in the reaction was not derived from that released into the bulk solution by sulfhydryl oxidase-catalyzed thiol oxidation. Further evidence for a direct transfer of the hydrogen peroxide moiety was obtained by addition of glutaraldehyde to a sulfhydryl oxidase/horseradish peroxidase/N-acetylcysteine mixture. Size exclusion chromatography revealed the formation of a high-molecular-weight species with peroxidase activity, which was completely resolved from native horseradish peroxidase. Formation of this species was absolutely dependent on the presence of both the cysteine-containing substrate and sulfhydryl oxidase. The observed enhancement of sulfhydryl oxidase catalytic activity by the addition of horseradish peroxidase supports a bi uni ping-pong mechanism proposed previously for sulfhydryl oxidase.
Acetylcysteine↗
A continuous spectrophotometric assay for monoamine oxidase and related enzymes in tissue homogenates.
A continuous, peroxidase-linked spectrophotometric assay is described which is suitable for measuring monoamine and diamine oxidase and semicarbazide-sensitive amine oxidase activities in tissue homogenates. In the assay, 4-aminoantipyrine is oxidized and then condenses with vanillic acid to give a red quinoneimine dye. The absorbance at 498 nm is proportional to the amount of hydrogen peroxide released in the amine oxidase reaction. The molar absorption coefficient of the dye at pH 7.6 was 4654 M-1 cm-1. The method is suitable for use with any amine oxidase substrate which has a higher oxidation-reduction potential than does 4-aminoantipyrine. Following preincubation of rat liver homogenates with selective monoamine oxidase (MAO)-A and -B inhibitors, kinetic constants were obtained for metabolism of the mixed substrate, p-tyramine. Inhibition of MAO in rat liver homogenates was also measured following administration of the antidepressant, phenelzine. This inexpensive assay which employs reagents with low toxicity can thus be used to determine the degree of inhibition of MAO elicited by potential antidepressant and anti-parkinsonian agents. Drugs, their metabolites, and environmental toxins can also be screened as possible amine oxidase substrates or inhibitors, and kinetic constants for turnover of novel substrates can be determined.
Absorption↗
Anti-oxidant enzymes in Cryptosporidium parvum oocysts.
Oocysts of Cryptosporidium parvum showed relatively low levels of SOD activity. The SOD which had a pI of 4.8 and an approximate molecular weight of 35 kDa appeared to be iron dependent. Catalase, glutathione transferase, glutathione reductase and glutathione peroxidase activity could not be detected, nor could trypanothione reductase. No NADH or NADPH oxidase activity could be detected, nor could peroxidase activity be demonstrated using o-dianisidine, guaiacol, NADPH or NADH as co-substrates. However, an NADPH-dependent H2O2 scavenging system was detected in the insoluble fraction.
Animals↗
Inhibition of lignin peroxidase-mediated oxidation activity by ethylenediamine tetraacetic acid and N-N-N'-N'-tetramethylenediamine.
The mineralization rate of LC-[1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane] (DDT) was reduced by 90% on the 18th day in fungal cultures of Phanerochaete chrysosporium in the presence of 8 mM ethylenediamine tetraacetic acid (EDTA). In the presence of 8 mM N-N-N'-N'-tetramethylenediamine (TEMED), the mineralization rate of 14C-DDT was reduced by 80%. In the presence of 2 mM or 10 mM EDTA, 95% inhibition of lignin peroxidase (LiP) mediated veratryl alcohol oxidase activity and 97% inhibition of LiP mediated iodide oxidase activity occurred. TEMED caused 79% inhibition of veratryl alcohol oxidase activity and 92% inhibition of iodide oxidase activity when the amount used was 2 mM and 10 mM, respectively. In the presence of Zn(II) with slight molar excess of the EDTA concentration, reversed the EDTA mediated non-competitive inhibition of LiP catalyzed veratryl alcohol or iodide oxidation, Zn(II) also reversed the inhibition of LiP catalyzed veratryl alcohol oxidase activity caused by chelators other than EDTA and TEMED. In addition to Zn(II), several other metal ions also relieved EDTA mediated inhibition of veratryl alcohol and iodide oxidase activity catalyzed by LiP. The ability of veratryl alcohol to inhibit iodide oxidation catalyzed by LiP showed that veratryl alcohol could inhibit LiP mediated iodide oxidase activity. Increasing the concentration of iodide was also shown to inhibit veratryl alcohol oxidation. Kinetic analysis showed that the reaction was competitive inhibition.
Alcohol Oxidoreductases↗
Enzymic determination of free fatty acids in serum.
We describe the enzymic determination of free fatty acids in serum with use of acyl-CoA synthetase (EC l.2.1.3), acyl-CoA oxidase (no EC no. assigned) and peroxidase (EC 1.11.1.7). The free fatty acids are activated by acyl-CoA synthetase in the presence of ATP and coenzyme A. The acyl-CoA formed is oxidized by acyl-CoA oxidase to enoyl-CoA with simultaneous production of hydrogen peroxide, which is oxidatively coupled with 4-amino-antipyrine and 2,4-dibromphenol in the presence of peroxidase to yield a product that absorbs maximally at 505 nm. Standard curves prepared with various kinds of fatty acids are linear to at least 2.0 mmol/L and are practically congruent. Ascorbic acid or bilirubin interferes slightly. Results by the present method correlate well with those by the colorimetric method involving 2-(2-thiozolylazo)-p-cresol (r = 0.98). Replicate analyses of standard solution and of two kinds of control sera demonstrated the following between-assay precision: mean absorbance 0.224 (SD 0.002), CV 0.85%; mean concentration, 326 (SD 7.3) and 1076 (SD 22.7) mumol/L, CV 2.24 and 2.11%, respectively.
Acyl Coenzyme A↗
Biochemical targets for antifungal azole derivatives: hypothesis on the mode of action.
The selective interaction of low concentrations of azole derivatives and other nitrogen heterocycles with cytochrome P-450 may be at the origin of the inhibition of ergosterol biosynthesis. From the depletion of ergosterol and the concomitant accumulation of 14 alpha-methylsterols, alterations in membrane functions, the synthesis and activity of membrane-bound enzymes, mitochondrial activities, and an uncoordinated activation of chitin synthase may result. Since chitin synthesis is more important in the hyphal form than in the budding form of C. albicans, the uncoordinated activation of chitin synthesis may be more trouble for the hyphal growth than for yeast budding. The assumption is made that from this difference the greater sensitivity of hyphal growth to azole antifungal agents may originate. It is also assumed that the higher degree of lipid unsaturation may be related to an inhibition of ergosterol biosynthesis. The inhibition of fatty acid desaturation and elongation induced by higher doses of miconazole and ketoconazole and the longer contact times might be related to interference with membrane fluidity, or it might due to chelation of the iron used in the oxidation reduction sequence during desaturation. The decreased availability of ergosterol and the accumulation of 14 alpha-methylsterols also may provide the environment needed to inactivate membrane-bound enzymes; e.g., cytochrome c peroxidase. However, it is still too speculative to correlate effects on membrane components with miconazole-induced changes in properties of all oxidases; e.g., the NADH-dependent, cyanide-insensitive oxidase. The accumulation of toxic concentrations of hydrogen peroxide, resulting from an increased NADH-oxidase activity and disappearance of the peroxidase and catalase activity, may contribute to the degeneration of subcellular structures. The complete disappearance of catalase observed at concentrations of miconazole greater than or equal to 10(-5) M may originate from direct effects on the cell. At these high concentrations reached only by topical application, direct membrane damage resulting from interaction of miconazole with lipids was observed. These direct interactions result in an inhibition of membrane-bound enzyme and mitochondrial activities and in leakage of intracellular components. The direct interactions were much less pronounced in cells treated with ketoconazole. This correlates with the smaller area occupied in the membrane per ketoconazole molecule (30 A2), compared with that occupied in the membrane per miconazole molecule (90 A2).(ABSTRACT TRUNCATED AT 400 WORDS)
Animals↗