Xanthine Dehydrogenase Activity Research Articles

Nitrogen excretion in Japanese quail during protein depletion and repletion.

The influence of protein depletion and repletion on the excretion of uric acid, urea, creatinine and free arginine as well as on the activities of xanthine dehydrogenase and arginase in Japanese quail was studied. Adult cockerels normally fed until the age of 42 days were kept on a protein free diet for 21 days and subsequently for 14 days on the previous normal diet. The control group was fed normal diet for the whole time. In 1-5 days intervals the enzyme activities and the N-metabolites in 24 hours excreta were determined. Protein free diet feeding evoked rapid (in term of 8-9 days) diminution of the excretion of N-metabolites, as well as of the xanthine dehydrogenase activity up to the fourth-tenth part of the initial values. The arginase activity was not influenced by this treatment. Body mass decreased by 36%. Returning to the normal protein feeding reverse the values of observed parameters to their initial levels within 8-9 days. It was concluded that changes in excreted N-metabolites caused by protein free feeding were rapid and recoverable by returning to normal diet.

Effect of adenine metabolites on survival of Drosophila melanogaster of low xanthine dehydrogenase activity

1. 1. Low xanthine dehydrogenase (LXD) mutant Drosophila melanogaster were fed 0.2% adenine for 7 generations, no adenine for the next 2 generations (relaxed) and 0.2% adenine again for the next 3 generations (rechallenged) to obtain adenine-resistant lines of Drosophila (LXD-adenine). Flies grown without adenine served as LXD-controls. 2. 2. Purines ranked as follows; adenine > adenosine > AMP > inosine > IMP in decreasing order of toxicity to LXD-adenine flies. 3. 3. Addition to ribose to 9N position, or phosphate or carboxy to 6C position of the purine ring alleviated the toxicity. 4. 4. More LXD-adenine offspring survived than did LXD-control offspring rechallenged with adenine.

Purification and substrate inactivation of xanthine dehydrogenase from Chlamydomonasreinhardtii

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 exhibited a specific activity of 5.7 units/ mg protein (turnover number = 1.9 · 10 3 min −1) and a remarkable instability at roo, temperature. Spectral properties were identical 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 provokrf enzymed reduction which was followed by inactivation. This irreversible inactivation also took place either under anerobic 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 inactivated 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 xathine incubation. We conclude that xanthine dehydrogenase inactivation by subtrate is due to an irreversible process affecting mainly molybdenum and that sequential andn uniterrupted electron flow from xanthine to NAD + is essential to maintain the enzyme in its active form.

Interferons induce xanthine dehydrogenase gene expression in L929 cells

Human interferon-alpha A/D (Bg/II) (IFN-alpha A/D) and mouse interferon-gamma (IFN-gamma) are shown to induce xanthine dehydrogenase (XD) mRNA in L929 fibroblastic cells. XD mRNA accumulation after IFN-alpha A/D treatment is relatively fast, being already evident after 4 h and reaching its maximum after 24 h. IFN-alpha A/D is active in inducing XD mRNA at 0.1 unit/ml and it is maximally active at 10(3) units/ml. The half-life of the XD message is unaffected by IFN-alpha A/D treatment, whereas the transcriptional activity of the XD gene and the concentrations of XD heterogeneous nuclear RNA are increased by 2- and 6-fold respectively. The effect of IFN-alpha A/D on XD mRNA is insensitive to cycloheximide, suggesting that protein synthesis de novo is not required. Experiments conducted with specific inhibitors suggest that protein kinase C, cyclic AMP and arachidonic acid metabolites derived from lipoxygenase or cyclooxygenase do not act as second-messenger molecules in the induction of XD mRNA by IFN-alpha A/D. XD mRNA is also induced in NIH3T3 fibroblastic cells, but not in F9 teratocarcinoma or B16 melanoma cells after treatment with IFN-alpha A/D. NIH3T3 are the only cells so far tested that have detectable XD and xanthine oxidase activities under basal conditions and after IFN-alpha A/D treatment, although their responsiveness to the cytokine is much less than that observed in L929 cells.

Use of rosy mutant strains of Drosophila melanogaster to probe the structure and function of xanthine dehydrogenase

The usefulness in structure/function studies of molybdenum-containing hydroxylases in work with rosy mutant strains of Drosophila melanogaster has been investigated. At least 23 such strains are available, each corresponding to a single known amino acid change in the xanthine dehydrogenase sequence. Sequence comparisons permit identification, with some certainty, of regions associated with the iron-sulphur centres and the pterin molybdenum cofactor of the enzyme. Procedures have been developed and rigorously tested for the assay in gel-filtered extracts of the flies, of different catalytic activities of xanthine dehydrogenase by the use of various oxidizing and reducing substrates. These methods have been applied to 11 different rosy mutant strains that map to different regions of the sequence. All the mutations studied cause characteristic activity changes in the enzyme. In general these are consistent with the accepted assignment of the cofactors to the different domains and with the known reactivities of the molybdenum, flavin and iron-sulphur centres. Most results are interpretable in terms of the mutation affecting electron transfer to or from one redox centre only. The activity data provide evidence that FAD and the NAD+/NADH binding sites are retained in mutants mapping to the flavin domain. Therefore, despite some indications from sequence comparisons, it is concluded that the structure of this domain of xanthine dehydrogenase cannot be directly related to that of other flavoproteins for which structural data are available. The data also indicate that the artificial electron acceptor phenazine methosulphate acts at the iron-sulphur centres and suggest that these centres may not be essential for electron transfer between molybdenum and flavin. The work emphasizes the importance of combined genetic and biochemical study of rosy mutant xanthine dehydrogenase variants in probing the structure and function of enzymes of this class.

Characterization of a rice (Oryza sativa L.) mutant deficient in the heme domain of nitrate reductase

Biochemical and genetical characterization of a rice nitrate reductase (NR)-deficient mutant, M819, which had been isolated as a chlorate-resistant mutant, was carried out. In M819, leaf NADH-NR activity was found to be about 10% of that of the wild-type cv 'Norin 8', while NADPH-NR activity was higher than that in the wild-type; FMNH2-NR and MV-NR activities were also 10% of those of the wild type; BPB-NR activity was higher than that of the wild type; and xanthine dehydrogenase activity was revealed to be present in both. These results suggest that the mutant line M819 lacks the functional heme domain of the NADH-NR polypeptide due to a point mutation or a small deletion within the coding region of the structural gene. Chlorate resistance in M819 was transmitted by a single recessive nuclear gene.

The contribution of vascular endothelial xanthine dehydrogenase/oxidase to oxygen-mediated cell injury

The conversion of xanthine dehydrogenase (XDH) to xanthine oxidase (XO) and the reaction of XO-derived partially reduced oxygen species (PROS) have been suggested to be important in diverse mechanisms of tissue pathophysiology, including oxygen toxicity. Bovine aortic endothelial cells expressed variable amounts of XDH and XO activity in culture. Xanthine dehydrogenase plus xanthine oxidase specific activity increased in dividing cells, peaked after achieving confluency, and decreased in postconfluent cells. Exposure of BAEC to hyperoxia (95% O 2; 5% CO 2) for 0–48 h caused no change in cell protein or DNA when compared to normoxic controls. Cell XDH + XO activity decreased 98% after 48 h of 95% O 2 exposure and decreased 68% after 48 h normoxia. During hyperoxia, the percentage of cell XDH + XO in the XO form increased to 100%, but was unchanged in air controls. Cell catalase activity was unaffected by hyperoxia and lactate dehydrogenase activity was minimally elevated. Hyperoxia resulted in enhanced cell detachment from monolayers, which increased 112% compared to controls. Release of DNA and preincorporated [8- 14C]adenine was also used to assess hyperoxic cell injury and did not significantly change in exposed cells. Pretreatment of cells with allopurinol for 1 h inhibited XDH + XO activity 100%, which could be reversed after oxidation of cell lysates with potassium ferricyanide (K 3Fe(CN) 6). After 48 h of culture in air with allopurinol, cell XDH + XO activity was enhanced when assayed after reversal of inhibition with K 3Fe(CN) 6, and cell detachment was decreased. In contrast, allopurinol treatment of cells 1 h prior to and during 48 h of hyperoxic exposure did not reduce cell damage. After K 3Fe(CN) 6 oxidation, XDH + XO activity was undetectable in hyperoxic cell lysates. Thus, XO-derived PROS did not contribute to cell injury or inactivation of XDH + XO during hyperoxia. It is concluded that endogenous cell XO was not a significant source of reactive oxygen species during hyperoxia and contributes only minimally to net cell production of O 2 − and H 2O 2 during normoxia.

Responses of vascular endothelial oxidant metabolism to lipopolysaccharide and tumor necrosis factor-α

Quantification of intracellular and extracellular levels and production rates of reactive oxygen species is crucial to understanding their contribution to tissue pathophysiology. We measured basal rates of oxidant production and the activity of xanthine oxidase, proposed to be a key source of O 2 − and H 2O 2, in endothelial cells. Then we examined the influence of tumor necrosis factor-α and lipopolysaccharide on endothelial cell oxidant metabolism, in response to the proposal that these inflammatory mediators initiate vascular injury in part by stimulating endothelial xanthine oxidase-mediated production of O 2 − and H 2O 2. We determined a basal intracellular H 2O 2 concentration of 32.8 ± 10.7 p m in cultured bovine aortic endothelial cells by kinetic analysis of aminotriazole-mediated inactivation of endogenous catalase. Catalase activity was 5.72 ± 1.61 U/mg cell protein and glutathione peroxidase activity was much lower, 8.13 ± 3.79 mU/ mg protein. Only 0.48 ± 0.18% of total glucose metabolism occurred via the pentose phosphate pathway. The rate of extracellular H 2O 2 release was 75 ± 12 pmol · min −1 · mg cell protein −1. Intracellular xanthine dehydrogenase/oxidase activity determined by pterin oxidation was 2.32 ± 0.75 μU/mg with 47.1 ± 11.7% in the oxidase form. Intracellular purine levels of 1.19 ±1.04 nmol hypoxanthine/mg protein, 0.13 ± 0.17 nmol xanthine/mg protein, and undetectable uric acid were consistent with a low activity of xanthine dehydrogenase/ oxidase. Exposure of endothelial cells to 1000 U/ml tumor necrosis factor (TNF) or 1 μg/ml lipopolysaccharide (LPS) for 1–12 h did not alter basal endothelial cell oxidant production or xanthine dehydrogenase/oxidase activity. These results do not support a casual role for H 2O 2 in the direct endothelial toxicity of TNF and LPS.

Identification of two tungstate-sensitive molybdenum cofactor mutants, chl2 and chl7, of Arabidopsis thaliana.

The characterization of mutants that are resistant to the herbicide chlorate has greatly increased our understanding of the structure and function of the genes required for the assimilation of nitrate. Hundreds of chlorate-resistant mutants have been identified in plants, and almost all have been found to be defective in nitrate reduction due to mutations in either nitrate reductase (NR) structural genes or genes required for the synthesis of the NR cofactor molybdenum-pterin (MoCo). The cholorate-resistant mutant of Arabidopsis thaliana, chl2, is also impaired in nitrate reduction, but the defect responsible for this phenotype has yet to be explained. chl2 plants have low levels of NR activity, yet the map position of the chl2 mutation is clearly distinct from that of the two NR structural genes that have been identified in Arabidopsis. In addition, chl2 plants are not thought to be defective in MoCo, as they have near wild-type levels of xanthine dehydrogenase activity, which has been used as a measure of MoCo in other organisms. These results suggest that chl2 may be a NR regulatory mutant. We have examined chl2 plants and have found that they have as much NR (NIA2) mRNA as wild type a variable but often reduced level of NR protein, and one-eighth the NR activity of wild-type plants. It is difficult to explain these results by a simple regulatory model; therefore, we reexamined the MoCo levels in chl2 plants using a sensitive, specific assay for MoCo: complementation of Neurospora MoCo mutant extracts.(ABSTRACT TRUNCATED AT 250 WORDS)

Hypoxia injures endothelial cells by increasing endogenous xanthine oxidase activity.

Exposure to decreasing oxygen tensions progressively increased xanthine dehydrogenase (XD) and xanthine oxidase (XO) activities over 48 hr in cultured pulmonary artery endothelial cells (EC) without altering XD/XO ratios. Increases in XD and XO activity in EC induced by hypoxia were associated upon reoxygenation with increased (P less than 0.05) extracellular superoxide anion (O2-.) levels that were inhibited by treatment with XO inhibitors (tungsten, allopurinol) or an anion-channel blocker (4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid). EC monolayers subjected to hypoxia/reoxygenation also leaked more preloaded 51Cr, were more adherent to neutrophils, and permitted greater albumin transit than control monolayers. Treatment with tungsten, allopurinol, and/or superoxide dismutase decreased (P less than 0.05) 51Cr release, neutrophil adherence, and albumin transit in EC monolayers exposed to hypoxia/reoxygenation. We conclude that prolonged hypoxia increases both XO and XD activity in EC and may predispose the endothelium to oxidative and inflammatory damage.

Pulmonary microvascular endothelial cells constitutively release xanthine oxidase

The generation of oxidants in reperfused ischemic tissues by xanthine oxidase (XO) may contribute to tissue damage. We exposed bovine pulmonary microvascular endothelial (BPMVE) cells to hypoxia and subsequent reoxygenation and examined alterations in intracellular and extracellular XO activities. BPMVE cells incubated 24 h under hypoxic conditions (<1% O 2) showed a twofold increase in intracellular xanthine dehydrogenase activity and a smaller increase in intracellular XO activity compared to normoxic BPMVE. Both normoxic and hypoxic BPMVE cells constitutively released XO activity into their culture media. Incubation of hypoxic or normoxic BPMVE cells with oxygenated medium (95% O 2) stimulated the release of XO activity into the extracellular medium within 5 min. The XO activity could not be detected in the oxygenated medium after 60 min incubation with 95% O 2. These results indicate that endothelial cells in culture constitutively release XO and that oxygenation rapidly enhances XO release. The released XO activity may play an important role in generation of oxidants in the extracellular milieu during reperfusion.

Purine metabolism and B-lymphocyte development in the chicken bursa of fabricius

The activity of three enzymes involved in the salvage pathway of purine nucleosides—purine nucleoside phosphorylase (PNP), xanthine dehydrogenase (XDH), and hypoxanthine-guanine phosphoribosyl transferase (HGPRT)—was investigated in cellular fractions of the chicken bursa of Fabricius differentially enriched in epithelial cells or lymphocytes. Markedly increasing levels of PNP and XDH were observed along with the enrichment in epithelial cells together with a slight, though significant, decrease in HGPRT activity. By contrast, a dramatic fall in PNP and XDH activities was detected along with the enrichment in lymphocytes together with a slight, though significant, increase in HGPRT activity. This sharply different distribution of the three enzymes, all sharing hypoxanthine as a substrate, clearly indicates that lymphocytes preferentially channel hypoxanthine into the salvage and interconversion pathways, phosphorylating it to IMP, while epithelial cells rapidly catabolize such a purine base to uric acid. Moreover, epithelial cells, unlike lymphocytes, are able to retain high intracellular levels of both hypoxanthine and inosine. These results support the possibility that epithelial cells contribute to the normal development of bursal lymphocytes by supplying such actively proliferating cells with purine rings and at the same time by preventing them from accumulating potentially toxic high levels of purine nucleotides being able to rapidly eliminate excess hypoxanthine as uric acid from the bursa environment into the bloodstream.

Involvement of histidine residues in catalytic activity of xanthine dehydrogenase from hen liver

1. 1. Modification of histidine residue(s) of xanthine dehydrogenase from hen liver by DEP and photooxidation results in loss of the ability to transfer electrons from xanthine to NAD + and also from NADH to 2,6-dichlorophenolindophenol (DCIP). 2. 2. The kinetics of inactivation suggest that carbethoxylation of more than one histidyl residue in the enzyme may be responsible for the inactivation.

Xanthine oxidase and dehydrogenase activities in rat ovarian tissues

The production of reactive oxygen species in the ovary is rapidly inducible, but the nature of the generator is unknown. One possibility is xanthine oxidase (XO), an enzyme that produces superoxide in the presence of hypoxanthine (or xanthine) and oxygen. The objective of the present studies was to measure levels of XO in follicular and luteal tissue to determine whether XO may be a source of reactive oxygen species in the rat ovary. Ovarian levels of XO were about one-fifth of that seen in the liver and adrenal, and XO levels were about one-third of xanthine dehydrogenase (XDH). Preovulatory ovarian levels of XO activity were unchanged after induction of ovulation with gonadotropin and in follicles incubated with gonadotropin. Luteal XO activity was not changed during natural or prostaglandin F2 alpha (PGF2 alpha)-induced luteolysis. Allopurinol, an inhibitor of XO, did not inhibit ovulation or PGF2 alpha-induced luteal regression. Finally, neither catalase and superoxide dismutase nor oxypurinol altered luteal cell function in the presence of hypoxanthine. Thus, while XO is present in the ovary, it does not appear that it is a major source of reactive oxygen species in this organ.

Change of xanthine dehydrogenase and xanthine oxidase activities in rat brain following complete ischaemia.

We studied the activities of xanthine dehydrogenase and xanthine oxidase in rat forebrain after complete ischaemia. Complete ischaemia was induced by decapitation after transcardiac infusion with saline. The activities of xanthine dehydrogenase and xanthine oxidase immediately after ischaemia were 93.3 +/- 38.7 and 18.8 +/- 7.7 microU/mg protein, respectively, and at 24 h after ischaemia were 183.5 +/- 75.1 and 60.8 +/- 15.2 microU/mg protein, respectively. The ratios of xanthine dehydrogenase/xanthine oxidase immediately and 24 h after ischaemia were 5.04 +/- 1.03 and 3.04 +/- 0.99, respectively. These data indicate that xanthine dehydrogenase and xanthine oxidase activities were maintained even 24 h after complete ischaemia. Conversion of xanthine dehydrogenase to xanthine oxidase proceeds slowly during complete ischaemia.

Xanthine Oxidase/Dehydrogenase Activity in Intact Cultured Cells (In Situ Analysis)

The measured ratio of xanthine oxidase activity to the total activity of xanthine oxidase and dehydrogenase showed higher values in intact cells than when similar cells were homogenized. The total activity was the same for both systems. The xanthine oxidase ratio was 90, 60, 50, 50, 60% in V79, RIF/Ha3, SCC7, KHT intact cells and freshly extracted murine peritoneal macrophages respectively while the corresponding ratios measured were 25, 40, 38, 35, 22% when the cells were lysed by homogenization. Superoxide radical O2-. production by addition of xanthine to intact or homogenized cells to activate intracellular xanthine oxidase was higher in intact than homogenized cells. Homogenization of cells and tissues in the presence of dithioerythritol (DTE) can evidently lead to a considerable under-estimation of the xanthine oxidase ratio. The effect of hypoxia on cells has also been examined.

Regulation of xanthine dehydrogenase and xanthine oxidase activity and gene expression in cultured rat pulmonary endothelial cells.

The central importance of xanthine dehydrogenase (XDH) and xanthine oxidase (XO) in the pathobiochemistry of a number of clinical disorders underscores the need for a comprehensive understanding of the regulation of their expression. This study was undertaken to examine the effects of cytokines on XDH/XO activity and gene expression in pulmonary endothelial cells. The results indicate that IFN-gamma is a potent inducer of XDH/XO activity in rat lung endothelial cells derived from both the microvasculature (LMVC) and the pulmonary artery. In contrast, interferon-alpha/beta, tumor necrosis factor-alpha, interleukin-1 or -6, lipopolysaccharide and phorbol myristate acetate have no demonstrable effect. The increase in XDH/XO activity requires new protein synthesis. By Northern analysis, IFN-gamma markedly increases the level of the 5.0-kb XDH/XO mRNA in LMVC. The increase is due, in part, to increased transcription rate of the XDH/XO gene. Transcriptional activation does not require new protein synthesis. The physiologic relevance of these observations was evaluated by administering IFN-gamma to rats. Intraperitoneal administration leads to an increased XDH/XO activity and XDH/XO mRNA level in rat lungs. In sum, IFN-gamma is a potent and biologically relevant inducer of XDH/XO expression; the major site of upregulation occurs at the transcriptional level.

2,6-dichlorophenolindophenol is a competitive inhibitor for xanthine oxidase and is therefore not usable as an electron acceptor in the fluorometric assay

Xanthine oxidase has been recognized as an important source of oxygen free radicals in ischemia-reperfusion injury. In order to study enzyme in biological tissues, the conversion of of pterin (2-amino-4-hydroxypteridine) of isoxanthopterin provides the basis for a very sensitive fluorometric assay. Xanthine oxidase is typically assayed in the presence of pterin only, while an electron acceptor which replaces NAD + is used to determine the combined xanthine dehydrogenase plus xanthine oxidase activity. 2,6-Dichlorophenol-indophenol has been used as an electron acceptor in this aasay. However, it was found in this study that it acts as an effective competitive inhibitor for xanthine oxidase. We conclude that methylene blue is the electron acceptor of choice in the fluorometric assays for xanthine oxidase.

Role of oxygen radicals in tourniquet-related ischemia-reperfusion injury of human patients

In the current study we evaluated effluent blood from extremities of human patients undergoing reconstructive surgical treatment which is routinely accompanied by upper extremity exsanguination and application of a tourniquet. Following tourniquet release (reperfusion), there were immediate increases in the plasma levels of xanthine oxidase activity, uric acid, and histamine. Xanthine dehydrogenase activity was not detectable. Plasma also contained products consistent with the formation of oxygen-derived free radicals, namely hemoglobin and fluorescent compounds. Our data indicate in humans that ischemia-reperfusion events are associated with the appearance of xanthine oxidase activity and its products in the plasma effluent.

Absence of xanthine oxidoreductase activity in human myocardium

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. 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. 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). 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. 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.