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PubMed1991

Distinction between Hypoxanthine and Xanthine Transport in Chlamydomonas reinhardtii.

Chlamydomonas reinhardtii cells consumed hypoxanthine and xanthine by means of active systems which promoted purine intracellular accumulation against a high concentration gradient. Both uptake and accumulation were also observed in mutant strains lacking xanthine dehydrogenase activity. Xanthine and hypoxanthine uptake systems exhibited very similar Michaelis constants for transport and pH values, and both systems were induced by either hypoxanthine or xanthine. However, they differed greatly in the length of the lag phase before uptake induction, which was longer for hypoxanthine than for xanthine. Cells grown on ammonium and transferred to hypoxanthine media consumed xanthine before hypoxanthine, whereas cells transferred to xanthine media did not take up hypoxanthine until 2 hours after commencing xanthine consumption. Metabolic and photosynthetic inhibitors such as 2,4-dinitrophenol, 3-(3,4-dichlorophenyl)-1,1-dimethyl urea, and carbonylcyanide m-chlorophenylhydrazone inhibited to a different extent the hypoxanthine and xanthine uptake. Similarly, N-ethylmaleimide abolished xanthine uptake but slightly affected that of hypoxanthine. Hypoxanthine consumption was inhibited by adenine and guanine whereas that of xanthine was inhibited only by urate. We conclude that hypoxanthine and xanthine in C. reinhardtii are taken up by different active transport systems which work independently of the intracellular enzymatic oxidation of these purines.

Journal Article↗
PubMed1991

Absence of xanthine oxidoreductase activity in human myocardium.

STUDY OBJECTIVE: The aim was to examine whether or not xanthine oxidase activity may be a significant source of oxygen derived free radicals in the human heart. DESIGN: Xanthine oxidoreductase activity of human myocardium was assayed in vitro. In addition, tests were performed to assess whether or not endogenous inhibitors of the enzyme were present in myocardial homogenates. The enzyme assay was based on high performance liquid chromatography with electrochemical and/or radiochemical detection of hypoxanthine, xanthine, and urate. SUBJECTS: Measurements were done on (a) isolated perfused rat myocardia and (b) left ventricular needle biopsies and papillary muscles obtained during elective cardiac surgery (chiefly aortic and/or mitral valve replacement and aortocoronary bypass) (n = 105 patients). MEASUREMENTS AND MAIN RESULTS: Homogenisation of human papillary muscles in buffer caused significant accumulation of hypoxanthine but not xanthine or urate. In addition, during incubation of crude myocardial homogenates with exogenous xanthine or hypoxanthine in the presence of NAD+ and/or O2 no production of urate was detected. Likewise, following aerobic incubation of papillary muscle homogenates with 14C-hypoxanthine neither 14C-xanthine nor 14C-urate were formed. Absence of xanthine oxidising activity was also observed with human papillary muscle extracts that were subjected to either ultrafiltration or gel filtration. In contrast, the rat heart was found to contain abundant xanthine oxidoreductase activity. The rat heart enzyme was inhibited by both allopurinol and oxypurinol but remained active when mixed with human papillary muscle homogenates. CONCLUSIONS: These findings show absence of xanthine oxidase and xanthine dehydrogenase activities in human myocardium, indicating that xanthine oxidase is not a source of oxygen derived free radicals in the human heart.

Adolescent↗
PubMed1993

Myocardial xanthine oxidoreductase activity in hypertensive and hypercholesterolemic rats.

In several species, xanthine oxidoreductase activity seems to be a major source of free radicals in myocardial tissue. Its activity changes during development and aging, at least in the rat heart. Hardly any data are available about its activity in two important diseases, hypertension and hypercholesterolemia, in which the production of free radicals induced by xanthine oxidoreductase activity could play a role. Therefore we measured the activity of xanthine oxidase and dehydrogenase in myocardial tissue of spontaneously hypertensive. Wistar (control hypertensive), Yoshida (hypercholesterolemic) and Brown Norway (control hypercholesterolemic) rats of various ages. Cytosolic fractions were incubated at 30 degrees C, pH 8.3, with 60 microM xanthine, and the formation of urate was measured with high performance liquid chromatography. In the Wistar group, xanthine oxidoreductase activity was relatively constant during aging (about 1.8 U/g protein). In the hypertensive group, the activity increased gradually from 1.7 to 2.3 U/g at 18 months (p < 0.05 compared with Wistar at 18 months). Xanthine oxidase was about twice as high in both groups at 18 months (p < 0.001 compared with 2 and 6 months). The ratio of xanthine dehydrogenase to xanthine oxidase had decreased 42% at this age (p < 0.001). In the Yoshida and Brown Norway groups, xanthine oxidoreductase activity was similar, with a peak at 6 months. These data suggest that the hypercholesterolemic state does not influence xanthine oxidoreductase activity. In contrast, in hypertrophied myocardium, xanthine oxidoreductase activity was higher than in the control, suggesting a different potential for free-radical generation.(ABSTRACT TRUNCATED AT 250 WORDS)

Aging↗
PubMed1990

Xanthine oxidoreductase activity in perfused hearts of various species, including humans.

Oxygen free radicals generated by xanthine oxidase have been implicated in cardiac damage. The activity of xanthine oxidase/reductase in adult rat heart is considerable. Its assay gives controversial results for other species, for example, rabbits and humans. Therefore, we perfused isolated hearts of various species, including explanted human hearts, to measure the conversion of exogenous hypoxanthine to xanthine and urate. We assayed these purines with high-performance liquid chromatography. The apparent xanthine oxidoreductase activities, calculated as release of xanthine plus 2x urate, were (milliunits per gram wet weight, mean +/- SEM) mice 33 +/- 3 (n = 5), rats 28.5 +/- 1.4 (n = 9), guinea pigs 14.4 +/- 1.0 (n = 5), rabbits 0.59 +/- 0.09 (n = 5), pigs less than 0.1 (n = 6), humans 0.31 +/- 0.04 (n = 7), and cows 3.7 +/- 0.8 (n = 4). In rabbit heart the conversion of hypoxanthine to xanthine was slow, and that of xanthine to urate was even slower. On the other hand, guinea pig and human heart released little xanthine, indicating that xanthine breakdown exceeds its formation. We conclude that isolated perfused mouse, rat, guinea pig, and also bovine hearts show considerable xanthine oxidoreductase activity, contrasting rabbit, porcine, and diseased human hearts.

Animals↗
PubMed1982

Quantitation of rat liver xanthine oxidase by radioimmunoassay. A mechanism for sex-specific differences.

To further delineate the mechanism responsible for the differences in xanthine oxidase activity in male and female Sprague-Dawley rats, a sensitive and specific radioimmunoassay (RIA) was developed for the measurement of hepatic xanthine oxidase. The RIA could detect as little as 5 mg of liver enzyme. Specificity of the RIA was confirmed by 1) Ouchterlony double immuno-diffusion in which a single precipitin band exhibited xanthine oxidase activity, when crude liver homogenate and an enzyme-specific stain were used; 2) parallelism between purified 125I-labeled xanthine oxidase and serial dilutions of crude liver homogenate; 3) a linear correlation between xanthine oxidase activity and the level of enzyme protein; and 4) a single protein band coincident with purified xanthine oxidase, when an immunoprecipitate prepared from antisera and crude liver homogenate was analyzed on sodium dodecyl sulfate (SDS) polyacrylamide gels. Whether xanthine oxidase activity was assayed in the absence of nicotinamide adenine dinucleotide (NAD+) (oxidase form) or in the presence of NAD+ (dehydrogenase), male values were consistently higher, and both forms of the enzyme correlated significantly with each other. When purified to homogeneity, neither form of the enzyme was appreciably affected by 17 beta-estradiol or testosterone propionate. When the RIA was employed, levels of hepatic xanthine oxidase were significantly greater in male than in female rats. We concluded from these data that increased xanthine oxidase activity in the male corresponds to a greater quantitative complement of xanthine oxidase protein. Furthermore, lower xanthine oxidase activity in the female cannot be explained by immunologically cross-reactive material without enzyme activity nor by a direct sex-steroid enzyme interaction.

Animals↗
PubMed1976

Oxidation of selected pteridine derivatives by mamalian liver xanthine oxidase and aldehyde oxidase.

Considerable information is available concerning the oxidation of pteridine derivatives by bovine milk xanthine oxidase, but few investigations have been carried out on the oxidation of such compounds by mammalian liver xanthine oxidase and the related aldehyde oxidase. Xanthine oxidase, obtained from rat liver, oxidizes a variety of substituted amino- and hydroxypteridines in a manner identical to that previously observed for milk xanthine oxidase. For example, 2-aminopteridine and its 4- and 7-hydroxy derivatives were oxidized efficiently to 2-amino-4,7-dihydroxypteridine (isoxanthopterin) by the rat liver enzyme, and 4-aminopteridine and its 2- and 7-hydroxy derivatives were oxidized to 4-amino-2,7-dihydroxypteridine.4-Hydroxypteridine and the isomeric 2- and 7-hydroxypteridines were oxidized by rat liver xanthine oxidase to 2,4,7-trihydroxypteridine. Rabbit liver aldehyde oxidase, but not rat liver xanthine oxidase, was able to catalyze the oxidation in position 7 of 2,4-diaminopteridine and its 6-methyl and 6-hydroxymethyl derivatives. 2-Aminopteridine and 4-aminopteridine were both oxidized to the corresponding 7-hydroxy derivatives in the aldehyde oxidase system; 2-amino-4-hydroxypteridine appeared to be a minor product in the oxidation of 2-aminopteridine by rabbit liver aldehyde oxidase. Both aldehyde oxidase and xanthine oxidase were able to catalyze the oxidation of 2-amino-6,7-disubstituted pteridines to the corresponding 4-hydroxy derivatives; 4-hydroxy-6,7-disubstituted pteridines were oxidized in position 2 by both enzymes. 4-Amino-6,7-disubstituted pteridines were not oxidized by either enzyme. 2-Amino-4-methylpteridine was oxidized in position 7 by aldehyde oxidase but was not an effective substrate for xanthine oxidase; 2-hydroxypteridine and 7-hydroxypteridine were not oxidized to a detectably extent by aldehyde oxidase. All oxidations mediated by xanthine oxidase were strongly inhibited by allopurinol (4-hydroxypyrazolo[3,4-d]pyrimidine), and all oxidations mediated by aldehyde oxidase were inhibited by menadione (2-methyl-1,4-naphthoquinone). Rat liver xanthine oxidase and, to a lesser extent, rabbit liver aldehyde oxidase were inhibited by 4-chloro-6,7-dimethylpteridine; 2-amino-3-pyrazinecarboxylic acid inhibited xanthine oxidase but not aldehyde oxidase. The oxidations of 2- and 4-aminopteridines by aldehyde oxidase resulted in concomitant reduction of cytochrome c.

Aldehyde Oxidoreductases↗
PubMedDate not supplied

Arrhythmias and infarction in the ischemic pig heart are not mediated by xanthine oxidase-derived free oxygen radicals.

Xanthine oxidase activities of pig myocardium and blood during and following myocardial ischemia were measured using HPLC, and electrochemical detection of hypoxanthine, xanthine and uric acid. Myocardial ischemia was produced by occluding the anterior descending coronary artery two-thirds of the way from its origin. There was no accumulation of either xanthine or urate in the ischemic pig myocardium during occlusion periods of 90 min, but there was a substantial accumulation of hypoxanthine. Similarly, there was no increase in myocardial xanthine or urate during the 30 min reperfusion following coronary artery occlusion periods of 15, 30, 60 or 90 min. Following in vitro incubation at pH 8 of myocardial homogenates or blood with either hypoxanthine or xanthine and NAD, no urate production was detectable. In contrast, significant amounts of xanthine and/or urate were produced, following addition of xanthine oxidase to the reaction mixtures. Additional in vitro experiments showed that the following pig tissues were lacking xanthine oxidase activity: left and right atrial appendage, left and right ventricle, interventricular septum, anterior descending and circumflex coronary arteries, ascending aorta, lung, and blood. Large amounts of xanthine oxidase (9.3 +/- 1.8 SEM mU/g wet weight, n = 7) were found in pig liver. In the ischemic pig heart, transmural infarction developed within 60 min of ischemia. Ventricular arrhythmias and fibrillation occurred most frequently within 45 min of ischemia and within seconds after reperfusion. These results showed that the pig heart and blood were xanthine oxidase deficient, suggesting that xanthine oxidase-derived free oxygen radicals were not involved in the cytotoxic and arrhythmogenic effects brought about by myocardial ischemia and/or reperfusion in the pig.

Animals↗
PubMed1982

Characteristics of membrane-bound and soluble forms of xanthine oxidase from milk and endothelial cells of capillaries.

Xanthine oxidase (xanthine:O2 oxidoreductase, EC 1.2.3.2) was purified from bovine milk lipid globules to electrophoretic homogeneity (Mr 155,000) and antibodies were raised against it in rabbits. By immunolocalization techniques, the xanthine oxidase antigen was detected in milk lipid globules and mammary gland epithelium, but also in capillary endothelium from various tissues, including liver, lung and intestine. These findings were paralleled by measurements of xanthine oxidase activities in the tissues, both in a membrane-associated and a soluble form. Addition of hypoxanthine to fractions containing native xanthine oxidase did not promote lipid peroxidation, in contrast to the widely used in vitro system for lipid peroxidation which involves addition of xanthine oxidase preparations. Extraction with buffers of high ionic strength and with nonionic detergents removed only part of the enzyme from the membranes. Immunoprecipitates from the soluble supernatant fractions, using anti-xanthine oxidase IgG, were enriched in the Mr 155,000 polypeptide. Patterns of proteolytic cleavage products of the xanthine oxidase monomer from capillaries and milk lipid globules were similar but not identical. Immunoprecipitates from soluble fractions of milk lipid globules and tissues were enriched in both xanthine oxidase and NADH-cytochrome c reductase activities. Electrophoretic separation of proteins from milk lipid globule membranes under non-denaturing conditions revealed a close correlation of xanthine oxidase and part of the NADH-cytochrome c reductase activity, but showed different activity profiles of NADH-ferricyanide reductase and xanthine oxidase.

Adipose Tissue↗
PubMed1998

Xanthine dehydrogenase from the phototrophic purple bacterium Rhodobacter capsulatus is more similar to its eukaryotic counterparts than to prokaryotic molybdenum enzymes.

Fourteen Rhodobacter capsulatus mutants unable to grow with xanthine as sole nitrogen source were isolated by random Tn5 mutagenesis. Five of these Tn5 insertions were mapped within two adjacent chromosomal EcoRI fragments hybridizing to oligonucleotides synthesized according to conserved amino acid sequences of eukaryotic xanthine dehydrogenases. DNA sequence analysis of this region revealed two open reading frames, designated xdhA and xdhB, encoding xanthine dehydrogenase. The deduced amino acid sequence of XDHA contains binding sites for two [2Fe-2S] clusters and FAD, whereas XDHB is predicted to contain the molybdopterin cofactor. In contrast to R. capsulatus, these three cofactor binding sites reside within a single polypeptide chain in eukaryotic xanthine dehydrogenases. The amino acid sequence of xanthine dehydrogenase from R. capsulatus showed a higher degree of similarity to eukaryotic xanthine dehydrogenases than to the xanthine dehydrogenase-related aldehyde oxidoreductase from Desulphovibrio gigas. The expression of an xdhA-lacZ fusion was induced when hypoxanthine or xanthine was added as sole nitrogen source. Mutations in nifR1 (ntrC) and nifR4 (rpoN, encoding sigma54) had no influence on xdh gene expression. A putative activator sensing the availability of substrate seems to respond to xanthine but not to hypoxanthine. The transcriptional start site of xdhA was mapped by primer extension analysis. Comparison with known promoter elements revealed no significant homology. Xanthine dehydrogenase from R. capsulatus was purified to homogeneity. The enzyme consists of two subunits with molecular masses of 85 kDa and 50 kDa respectively. N-terminal amino acid sequencing of both subunits confirmed the predicted start codons. The molecular mass of the native enzyme was determined to be 275 kDa, indicating an alpha2beta2-subunit structure. Analysis of the molybdenum cofactor of xanthine dehydrogenase from R. capsulatus revealed that it contains the molybdopterin cofactor and not a molybdopterin dinucleotide derivative.

Amino Acid Sequence↗
PubMed1996

Characterization of xanthine dehydrogenase from the anaerobic bacterium Veillonella atypica and identification of a molybdopterin-cytosine-dinucleotide-containing molybdenum cofactor.

The molybdenum-containing iron-sulfur flavoprotein xanthine dehydrogenase from the anaerobic bacterium Veillonella atypica has been purified approximately 800-fold with a yield of approximately 40% and a specific activity of approximately 70 micromol ferricyanide reduced x min(-1) x mg protein(-1) with xanthine as electron donor, which corresponds to approximately 30 micromol xanthine oxidized x min(-1) x mg protein(-1) with methylene blue as electron acceptor. The 129-kDa enzyme was a non-covalent heterotrimer with large (82.4 kDa), medium (28.5 kDa) and small (18.4 kDa) subunits. The N-termini of the small and medium polypeptides of V. atypica xanthine dehydrogenase and the corresponding domains of eukaryotic xanthine dehydrogenases were similar, whereas the N-terminus of the large polypeptide was unrelated to eukaryotic xanthine dehydrogenases. The enzyme contained 0.86 atoms Mo, 1.75 atoms Fe, 1.61 atoms acid-labile sulfur and 0.68 molecules FAD/molecule, which corresponds to a 1:2.0:1.9:0.8 molar ratio. Acid hydrolysis revealed 0.95 mol CMP and 0.80 mol AMP/mol xanthine dehydrogenase. After treatment of the enzyme with iodoacetamide, di(carboxamidomethyl)molybdopterin cytosine dinucleotide was identified, which indicates that molybdopterin cytosine dinucleotide is the organic portion of the V. atypica xanthine dehydrogenase molybdenum cofactor. The enzyme and its molybdenum cofactor occurred in a 1:1 molar ratio. Xanthine dehydrogenases from eukaryotic sources are characterized by a domain structure and the presence of duplicate copies of two types of [2Fe-2S) clusters. In contrast, the xanthine dehydrogenase from V. atypica had a heterotrimeric subunit structure and a single [2Fe-2S] cluster. In addition, the enzyme indicates the presence of a molybdopterin dinucleotide as a constituent of a xanthine dehydrogenase molybdenum cofactor.

Amino Acid Sequence↗
PubMed1991

Inhibition of gonadotropin action and progesterone synthesis by xanthine oxidase in rat luteal cells.

Hydrogen peroxide produces marked antigonadotropic and lytic actions in luteal cells, but the effects of superoxide, the archetypal oxygen radical, are unknown. Xanthine oxidase generates superoxide, and the activity of this enzyme, and purine substrate, are increased under ischemia, such as that seen at luteal regression. We therefore examined the actions of xanthine oxidase on luteal cells to assess the effects of this enzyme and the superoxide anion on luteal function. Xanthine oxidase, in the presence of hypoxanthine (50 microM), produced marked inhibition of LH-sensitive cAMP and progesterone production with complete inhibition at 25 mU/ml and half-maximal inhibition at about 5 mU/ml. These antigonadotropic actions of xanthine oxidase were rapid with maximal effects within 5 min, followed several minutes later by substantial depletion of ATP. Heat, superoxide dismutase, and catalase or catalase alone abolished the actions of xanthine oxidase. While depletion of ATP by xanthine oxidase was prevented by 3-amino-benzamide, an inhibitor of DNA repair, inhibition of cAMP and progesterone production was still evident. Xanthine oxidase also inhibited progesterone synthesis stimulated by 8-bromo-cAMP. Isobutylmethylxanthine, a cAMP phosphodiesterase inhibitor, did not reverse the inhibition of cAMP accumulation by xanthine oxidase, and the enzyme had no effect on LH receptor binding activity. Since catalase reversed the effects of xanthine oxidase, we conclude that superoxide was rapidly dismuted to hydrogen peroxide and mediated the antigonadotropic and antisteroidogenic actions of xanthine oxidase in luteal cells. The sensitivity of luteal cells to xanthine oxidase raises the possibility that this enzyme may serve as a significant source of hydrogen peroxide in the corpus luteum.

1-Methyl-3-isobutylxanthine↗
PubMed1987

Xanthine oxidase is not responsible for reoxygenation injury in isolated-perfused rat heart.

The massive leakage of intracellular enzymes which occurs during reoxygenation of heart tissue after hypoxic or ischemic episodes has been suggested to result from the formation of oxygen radicals. One purported source of such radicals is the xanthine oxidase-mediated metabolism of hypoxanthine and xanthine. Xanthine oxidase (O form) has been suggested to be formed in vivo by limited proteolysis of xanthine dehydrogenase (D form) during the hypoxic period (Granger et al., Gastroenterology, 81, 22 (1981)). We measured the activities of xanthine oxidase in both fresh and isolated-perfused (Langendorff) rat heart tissue. Approximately 32% of the total xanthine oxidase was in the O form in fresh and isolated-perfused rat heart. This value was unchanged following 60 min of hypoxia and 30 minutes of reoxygenation. The infusion of 250 microM allopurinol throughout the perfusion completely inhibited xanthine oxidase activity but had no effect on the massive release of lactate dehydrogenase (LDH) into the coronary effluent upon reoxygenation of heart tissue subjected to 30 or 60 min of hypoxia. Protection from 30 min of hypoxia was also not obtained when rats were pretreated for 48 h with allopurinol at a dose of 30 mg/kg/day and perfused with allopurinol containing medium. Superoxide dismutase (50 units/ml), catalase (200 units/ml), or the antioxidant cyanidanol (100 microM) also had no effect on LDH release upon reoxygenation after 60 min of hypoxia. Xanthine oxidase activity was detected in a preparation enriched in cardiac endothelial cells while no allopurinol-inhibitable activity could be measured in purified isolated cardiomyocytes. It is concluded that xanthine dehydrogenase is not converted to xanthine oxidase in hypoxic tissue of the isolated perfused rat heart, and that the release of intracellular enzymes upon reoxygenation in this experimental model is mediated by factors other than reactive oxygen generated by xanthine oxidase.

Allopurinol↗
PubMed1978

Regulation, purification, and properties of xanthine dehydrogenase in Neurospora crassa.

Xanthine dehydrogenase (EC 1.2.1.37) is the first enzyme in the degradative pathway by which fungi convert purines to ammonia. In vivo, the activity is induced 6-fold by growth in uric acid. Hypoxanthine, xanthine, adenine, or guanine also induce enzyme activity but to a lesser degree. Immunoelectrophoresis using monospecific antibodies prepared against Neurospora crassa xanthine dehydrogenase shows that the induced increase in enzyme activity results from increased numbers of xanthine dehydrogenase molecules, presumably arising from de novo enzyme synthesis. Xanthine dehydrogenase has been purified to homogeneity by conventional methods followed by immunoabsorption to monospecific antibodies coupled to Sepharose 6B. Electrophoresis of purified xanthine dehydrogenase reveals a single protein band which also exhibits enzyme activity. The average specific activity of purified enzyme is 140 nmol of isoxanthopterine produced/min/mg. Xanthine dehydrogenase activity is substrate-inhibited by xanthine (0.14 mM), hypoxanthine (0.3 mM), and pterine (10 micron), is only slightly affected by metal binding agents such as KCN (6 mM), but is strongly inhibited by sulfhydryl reagents such as p-hydroxymercuribenzoate (2 micron). The molecular weight of xanthine dehydrogenase is 357,000 as calculated from a sedimentation coefficient of 11.8 S and a Stokes radius of 6.37 nm. Sodium dodecyl sulfate-gel electrophoresis of the enzyme reveals a single protein band having a molecular weight of 155,000. So the xanthine dehydrogenase protein appears to be a dimer. In contrast to xanthine dehydrogenases from animal sources which typically possess as prosthetic groups 2 FAD molecules, 2 molybdenum atoms, 8 atoms of iron, and 8 acid-labile sulfides, the Neurospora enzyme contains 2 FAD molecules, 1 molybdenum atom, 12 atoms of iron, and 14 eq of labile sulfide/molecule. The absorption spectrum of the enzyme shows maxima between 400 and 500 nm typical of a non-heme iron-containing flavoprotein.

Enzyme Induction↗
PubMed1990

Low-protein diet and xanthine-metabolising enzymes in adriamycin nephrosis.

Proteinuria and renal xanthine metabolising enzymes, xanthine oxidase and xanthine dehydrogenase, were evaluated in Adriamycin-treated rats fed standard (21% casein) and low-protein (6% casein) diets. In rats fed a standard diet Adriamycin was associated with increased activities in the kidney of xanthine oxidase and xanthine dehydrogenase and induced massive proteinuria. The pharmacological block of both enzymes by allopurinol and tungsten block of both enzymes by allopurinol and tungsten reduced proteinuria to one-third of the original levels. Rats fed a low-protein diet presented decreased levels of renal xanthine oxidase and xanthine dehydrogenase and were only slightly proteinuric. Finally, rats shifted from a low-protein diet to a normal one developed massive proteinuria in spite of normal or slightly decreased levels of renal xanthine oxidase and xanthine dehydrogenase. We conclude that a low-protein diet is effective in decreasing the levels of xanthine metabolising enzymes that are in part responsible for the renal damage due to Adriamycin. This is not however the unique mechanism by which the low-protein diet protects against the development of proteinuria in Adriamycin nephrosis; other factors must also be hypothesised.

Animals↗
PubMed2002

Global effects of xanthine oxidase stress on alveolar type II cells.

OBJECTIVE: To delineate biochemical details of graded xanthine oxidase stress toward cultured alveolar type II cells, particularly oxidant-mediated damage of type II cell nucleic acid, protein, and lipid, as an in vitro model of distant ischemia-reperfusion lung injury. DESIGN: In vitro injury model using native rat and immortalized mouse alveolar type II cells and exogenous xanthine oxidase. SETTING: Research laboratory. Measurement: Cultured type II cells were subjected to xanthine oxidase-derived reactive oxygen stress at variable concentrations and incubation times. Reduction of type II cell double-stranded DNA, inhibition of de novo phosphatidyl choline synthesis, enhancement of lipid peroxidation, and suppression of mitochondrial redox capacity were analyzed in relation to high-intensity (xanthine oxidase, 25 munits/mL) oxidant stress. Alterations in type II cell cellular glutathione-related antioxidant repertoire were assessed at both high-intensity and low-intensity (xanthine oxidase, 1 munits/mL) oxidant stress. MAIN RESULTS: High-intensity xanthine oxidase stress significantly increased type II cell DNA strand breakage, inhibited de novo phosphatidyl choline synthesis, diminished mitochondrial integrity, and enhanced lipid peroxidation in the absence of overt cytolysis. This injury was modulated with addition of exogenous glutathione peroxidase, or catalase/superoxide dismutase, but not glutathione or N-acetylcysteine. Although aspects of the glutathione antioxidant repertoire were similarly diminished with high-intensity xanthine oxidase stress, low-dose (long duration) xanthine oxidase stress augmented the activities of type II cell glutathione peroxidase and gamma-glutamyl transferase (the rate-limiting enzyme in glutathione synthesis). CONCLUSION: High-intensity xanthine oxidase stress (in vitro model of in vivo ischemia-reperfusion) may overwhelm type II cell antioxidant defenses and mediate oxidant injury to nucleic acid, protein, and lipid in the absence of cell lysis. Immortalized murine type II cells seem to appropriately model xanthine oxidase-mediated nucleic acid and protein injury of native rat type II cells. Exogenous glutathione peroxidase reduces oxidant injury in this in vitro model. Depending on magnitude (and possibly duration) of the xanthine oxidase stress, type II cell glutathione antioxidant elements may be diminished or enhanced.

Journal Article↗
PubMed1985

Intercellular nodule localization and nodule specificity of xanthine dehydrogenase in soybean.

The distribution of xanthine dehydrogenase throughout the soybean plant as well as the intercellular localization of xanthine dehydrogenase within soybean nodules was determined. Polyclonal antibodies against purified xanthine dehydrogenase were prepared and used in an enzymelinked immunosorbent assay to determine whether xanthine dehydrogenase is a nodule-specific protein. This immunological assay showed that xanthine dehydrogenase is present in far greater concentration in the nodule than in any other plant organ. Immunodiffusion tests showed that anti-soybean nodule xanthine dehydrogenase would cross-react with nodule crude extracts from the ureide producers, soybean, cowpea, and lima bean, but would not cross-react with those of the amide producers, alfalfa and lupine. A crude extract from pea nodules cross-reacted slightly with anti-soybean xanthine dehydrogenase. Anti-soybean xanthine dehydrogenase did not cross-react with buttermilk xanthine oxidase either by enzyme-linked immunosorbent assay or by immunodiffusion test.Fresh nodule sections from the ureide-producers, soybean, cowpea, and lima bean, all stained positively for xanthine dehydrogenase. The substrate-dependent stain was inhibited by allopurinol and was observed only in the infected nodule cells of these species. Nodules from the amideproducers, alfalfa and white lupine, did not stain for xanthine dehydrogenase.

Journal Article↗
PubMed1991

Effect of allyl alcohol on xanthine dehydrogenase activity in the perfused rat liver.

Xanthine oxidase has been implicated in the production of reactive oxygen species and cell injury produced by various toxic compounds. Since allyl alcohol injuries the liver by an oxygen-dependent mechanism, we examined the actions of this hepatotoxicant on the conversion of xanthine dehydrogenase into xanthine oxidase in perfused livers. A microassay for NAD(+)-dependent xanthine dehydrogenase, based on measuring the production of NADH fluorometrically under anaerobic conditions, was developed and used to examine the actions of allyl alcohol on this activity in periportal and pericentral regions of the liver lobule. The oxygen-dependent activity, xanthine oxidase, was monitored in whole liver homogenates by uric acid formation at 302 nm under aerobic conditions. Perfusion of the liver with allyl alcohol (350 microM) increased xanthine oxidase and decreased xanthine dehydrogenase in whole liver consistent with the hypothesis that allyl alcohol enhanced calcium-dependent proteolytic conversion of the NAD(+)-dependent to the O2-dependent form. Xanthine dehydrogenase was higher in pericentral than in periportal regions of the liver lobule and tended to decrease selectively in periportal zones of livers exposed to allyl alcohol. O2 uptake was stimulated transiently by allyl alcohol followed by subsequent inhibition of respiration. These results are consistent with the idea that conversion of NAD(+)-dependent xanthine dehydrogenase to xanthine oxidase is involved in the zone-specific hepatotoxicity of allyl alcohol.

1-Propanol↗
PubMed1989

The mobilization of ferritin iron by liver cytosol. A comparison of xanthine and NADH as reducing substrates.

Considerable evidence suggests that the release of iron from ferritin is a reductive process. A role in this process has been proposed for two hepatic enzymes, namely xanthine oxidoreductase and an NADH oxidoreductase. The abilities of xanthine and NADH to serve as a source of reducing power for the enzyme-mediated release of ferritin iron (ferrireductase activity) were compared with turkey liver and rat liver homogenates. The maximal velocity (Vmax.) for the reaction with NADH was 50 times greater than with xanthine; however, the substrate concentration required to achieve half-maximal velocity (Km) was 1000 times less with xanthine than with NADH. NADPH could be substituted for NADH with little loss in activity. Dicoumarol did not inhibit the reaction with NADH or NADPH, demonstrating that the ferrireductase activity with those substrates was not the result of the liver enzyme 'DT-diaphorase' [NAD(P)H dehydrogenase (quinone)]. A flavin nucleotide was required for ferrireductase activity with rat and turkey liver cytosol when xanthine, NADH or NADPH was used as the reducing substrate. FMN yielded twice the activity with NADH or NADPH, whereas FAD was twice as effective with xanthine as substrate. Kinetic comparisons, differences in lability and partial chromatographic resolution of the ferrireductase activities with the two types of reducing substrates strongly indicate that the ferrireductase activities with xanthine and NADH are catalysed by separate enzyme systems contained in liver cytosol. Complete inhibition by allopurinol of the ferrireductase activity endogenous to undialysed liver cytosol preparations and the ability of xanthine to restore equivalent activity to dialysed preparations indicate that the source of reducing power for the endogenous activity is xanthine. These studies suggest that xanthine, NADH or NADPH can serve as a source of reducing power for the enzyme-mediated reduction of ferritin iron, with a flavin nucleotide serving as the shuttle of electrons from the enzymes to the ferritin iron.

Animals↗