UNIPROT:P47989 - FACTA Search

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Query: UNIPROT:P47989 (xanthine oxidase)
8,633 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

During the aerobic conversion of xanthine to uric acid by xanthine oxidase, superoxide anion and hydrogen peroxide are produced along with the hydroxyl radical. Our studies demonstrate that washed human platelets incubated with xanthine and xanthine oxidase aggregated and released [14C]serotonin. Aggregation and release were dependent on the duration of exposure to xanthine oxidase as well as the concentration of enzyme. Both reactions were inhibited by the superoxide scavenger enzyme superoxide dismutase but not by catalase, or the free radical scavenger mannitol, suggesting that they were induced by superoxide anion. Superoxide-dependent release was inhibited by prior incubation of platelets with 1 mM EDTA, 1 micronM prostaglandin E1, or 1 mM dibutyryl cyclic AMP, but was unaffected by 1 mM acetylsalicylic acid or 1 micronM indomethacin. After prolonged incubation with xanthine and xanthine oxidase there was also efflux of up to 15% of intraplatelet 51Cr, a cytosol marker. This leakage was prevented by the addition of catalase to the media but not by superoxide dismutase. Incubation with xanthine and xanthine oxidase did not produce malonyldialdehyde, the three-carbon fatty acid fragment produced during prostaglandin endoperoxide synthesis and lipid peroxidation. Prior exposure of platelets to low fluxes of superoxide anion lowered the threshold for release by subsequent addition of thrombin, suggesting a synergistic effect. We conclude that superoxide-dependent aggregation and release may be a physiologically important method to modulate hemostatic reactions particularly in areas of inflammation or vessel injury which could have high local concentrations of superoxide anion.
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PMID:Enhancement of platelet function by superoxide anion. 19 66

The production of hydrogen peroxide was measured by following the oxidation of dichlorofluorescein (DCFH) entrapped into platelets. Resting platelets produced nanomolar quantities of DCF, which was proportional to the concentration of platelets and was steady during 1 h of incubation. A significant increase of basal DCF fluorescence was induced by stimuli namely thrombin, arachidonic acid, the Ca2+ ionophore A23187 and PMA. The effect of agonists has been also measured in the presence of 3-amino-1,2,4-triazole (AT) or N-ethylmaleimide (NEM), inhibitors of catalase and glutathione peroxidase, respectively. A further significant enhancement of DCF produced in stimulated platelets was detected only in the presence of NEM. A correlation was found between the increase in DCF and externally added hydrogen peroxide or the oxidizing species formed by xanthine oxidase plus acetaldehyde. The yield was not affected by superoxide dismutase and was higher in the presence of AT or NEM. A cooperative effect in the presence of both inhibitors was shown. Glutathione peroxidase plus glutathione diminished the level of DCF to basal levels.
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PMID:Generation of hydrogen peroxide in resting and activated platelets. 162 82

Rat serosal mast cells (MCs, 85-90% pure), obtained from peritoneal washing of Wistar albino rats, produced a significant amount of superoxide anions (O2.-) as measured by the increase in absorbance due to the reduction of ferricytochrome c; they were also able to generate a nitric oxide (NO)-like factor, as measured by two bioassay systems: i) inhibition of platelet aggregation and ii) stimulation of MCs guanylate cyclase. Incubation of MCs with human washed platelets resulted in an inhibition of thrombin-induced platelet aggregation which was proportional to cell number. The inhibitory activity of MCs was potentiated by substances which preserve NO (superoxide dismutase, SOD), and reversed by compounds which inactivate NO (oxyhaemoglobin, oxyHb) or which inhibit its synthesis (NG-monomethyl-L-arginine, MeArg). Mechanical stimulation of MCs produced a time-dependent increase in the levels of their cGMP but not cAMP; this increase was enhanced by E. coli lipopolysaccharide (LPS). NO generators such as sodium nitroprusside (NaNp) also augmented the levels of cGMP in MCs. NaNp inhibited in a dose-dependent manner the release of histamine evoked by compound 48/80 (0.5 microgram/ml), but not by the O2.--generating system (xanthine-xanthine oxidase), suggesting a bidirectional regulation of histamine release afforded by O2.- and NO.
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PMID:Mast cells as a source of superoxide anions and nitric oxide-like factor: relevance to histamine release. 172 22

If myocardial ischemia always results from an imbalance between the needs and supplies in oxygen of the myocardium cells, the physiopathology of this process seems today infinitely more complex than the mere diminution or interruption of the output in a coronary artery. The extension of atheromatous lesions, the platelets aggregation, thrombosis, the coronary spasm, the release of products from the arachidonic cascade, the reactivity of the vascular endothelium, the profibrinolytic activity of the tissues are many of the intricate factors inducing myocardial ischemia. Cellular alterations, of which some are triggered by the release of oxygenated free radicals, lead then to an irreversible necrosis. The medications used until now in the treatment of angina are oxygen scavengers and research goes on in this direction with vaso-dilators beta-blockers, prolonged action nitro-compounds (nicorandil) or nitro-compounds with an action reinforced by N-acetyl-cysteine, bradycardiac derivates of alinidine and the new calcium antagonists dihydropyridine. However, the new physiopathological concepts of ischemia have opened new directions for the research: products which modify the arachidonic cascade by increase of synthesis or release of PGI2 (nafazatrom, defibrotide), by inhibition of TXA2 synthesis or blocking of TXA2 receptors, and similar products of PGI2 (iloprost); thrombolytic agents more specific of thrombin (PTA) or fibrinolysis activators (defibrotide), and anticoagulants with extended action; chelating agents of oxygenated free radicals (peroxide dismutase, catalase, peroxidase) or xanthine oxidase inhibitors; platelets anti-aggregates like ticlopidine which blocks the platelets receptors to fibrinogen, or inhibitors of the synthesis of pro-aggregating agents.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:[Current therapeutic concepts in the treatment of myocardial ischemia. Current and future drugs]. 287 4

We investigated the effects of superoxide dismutase (SOD) and SOD linked to Ficoll (mol. wt = 400,000) on the changes in pulmonary transvascular fluid and protein exchange following pulmonary microembolism induced with alpha-thrombin. Studies were made in chronically prepared unanesthetized sheep with lung lymph fistulas. Control thrombin challenged sheep (n = 5) were compared to animals infused with SOD (the SOD-thrombin group, n = 5) or animals infused with SOD linked to high molecular weight Ficoll (the Ficoll-SOD-thrombin group, n = 6). The Ficoll-SOD-thrombin animals were also compared to animals infused with Ficoll alone (the Ficoll-thrombin group, n = 4). In the control-thrombin group, thrombin induced sustained increases in the pulmonary transvascular protein clearance (pulmonary lymph flow X lymph/plasma protein concentration ratio) and pulmonary vascular resistance (PVR). In the SOD-thrombin group, thrombin initially increased both pulmonary transvascular protein clearance and PVR; however, the later increases in protein clearance and PVR were blunted. The pulmonary reflection coefficients for total protein (sigma), a measure of vascular permeability to protein, decreased from a value of 0.70 +/- 0.03 in normal sheep to 0.60 +/- 0.01 following thrombin challenge (p less than 0.05) indicating an increase in lung vascular permeability. The sigma value in the SOD-treated animals was 0.70 +/- 0.02, indicating a protective effect of SOD. The infusion of the Ficoll-SOD complex also attenuated the increases in pulmonary transvascular protein clearance and PVR after thrombin. However, the infusion of Ficoll alone induced a similar protection. The lymph from the SOD-thrombin and Ficoll-SOD-thrombin groups prevented the reduction of ferricytochrome C by xanthine/xanthine oxidase, whereas, the lymph from the Ficoll-thrombin animals did not have this effect, indicating SOD activity was present in the animals receiving the enzyme but not in the group infused with Ficoll alone. Differences in the degree of intravascular coagulation could not explain the response to Ficoll since the decreases in fibrinogen concentration following the thrombin were similar in all the groups. Since Ficoll and related dextrans may modify neutrophil function, in particular neutrophil adherence to the endothelium, we examined the effects of Ficoll on neutrophil adherence. The results indicated that when Ficoll was added to the endothelial medium Ficoll reduced the increase adherence of neutrophils to the endothelial cell monolayer. Therefore, Ficoll as a carrier for SOD may provide a direct protection in models of lung vascular injury that are dependent on neutrophils.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Superoxide dismutase prevents the thrombin-induced increase in lung vascular permeability: role of superoxide in mediating the alterations in lung fluid balance. 302 59

Endothelial cell dysfunction is a key factor in oxidative stress-related pathology. Disruption of Ca2+ homeostasis is thought to be responsible for much of the endothelial cell dysfunction in oxidative stress. The expression of molecular chaperones (MC), which stabilize protein structures in normal and in stress conditions, reflects the Ca(2+)-dependent and -independent stress effects in the different cell compartments. By two-dimensional (2-D) gel electrophoresis combined with immunoblotting or microsequencing, we have identified 12 major MC in human umbilical vein endothelial cells (HUVEC): (i) the endoplasmic reticulum-located MC GRP78, GRP94, protein disulfide isomerase, and calreticulin; (ii) the mitochondrial MC HSP65 and GRP75; and (iii) the cytosolic/nuclear MC HSP27, HSC70, HSP70, HSP90, cyclophilin, and ubiquitin. To differentiate oxidative stress- and Ca(2+)-mediated effects, HUVEC were exposed to 1) xanthine oxidase plus hypoxanthine to generate oxidative stress, 2) ionomycin plus ethylene glycol-bis(beta-aminoethylether)-N,N,N', N'-tetraacetic acid (EGTA) to deplete intracellular Ca2+ stores, or 3) thrombin to increase cytosolic Ca2+. De novo protein synthesis after exposure was quantified by the incorporation of [35S]methionine. Image processing with the MELANIE system was used to create and compare the 2-D maps of [35S]methionine-labeled proteins under conditions 1)-3) with those of the controls. In a total of 24 2-D gels, 9 different MC were detected in at least 5 out 6 experimental replicates and were subjected to numeric analysis. The statistics showed a > 10% increase in GRP78 (p < 0.05), HSP27, cyclophilin, and ubiquitin after oxidative stress.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Effects of oxidative stress and Ca2+ agonists on molecular chaperones in human umbilical vein endothelial cells. 749 68

We compared the effects of phorbol 12-myristate 13-acetate (PMA) and thrombin with those of nonlytic concentrations of reactive oxygen species (ROS) generated by hypoxanthine (HX)-xanthine oxidase (XO) on the adhesion properties of human umbilical cord vein endothelial cells (HUVEC) to resting polymorphonuclear neutrophils (PMN). PMN adherence to HX-XO-treated HUVEC was increased approximately twofold to 2.5-fold relative to untreated HUVEC, both immediately and after 2 hours. It was not additive to that induced by PMA or thrombin stimulation of HUVEC. ROS-induced adherence was not due to platelet-activating factor (PAF) or P-selectin expression, as it was neither antagonized by BN52021 (PAF receptor antagonist) nor inhibited by anti-P-selectin monoclonal antibody (MoAb), contrary to the increased adhesion of PMA- and thrombin-stimulated HUVEC. PMN preincubated with mannose-6-P or N-acetylneuraminic acid (sialic acid), but not mannose or galactose-6-P, showed reduced adherence to ROS-treated HUVEC, suggesting that carbohydrate molecules were expressed on the latter and served as the ligand for the PMN L-selectin. Intercellular adhesion molecule (ICAM-1), constitutively present on the surface of resting HUVEC, was involved in the PMN adherence to ROS-treated HUVEC, since this adherence was inhibited by anti-ICAM-1, anti-CD11a, anti-CD11b, and anti-CD18 MoAbs. A non-CD18, non-ICAM-1-dependent mechanism is also involved in this adherence, since effects of these MoAbs were not additive; moreover, combinations of anti-CD18 and anti-ICAM-1 MoAbs with mannose-6-P and sialic acid completely inhibited PMN adherence. The increased binding of PMN to HX-XO-exposed HUVEC observed here involved IC-AM-1, but was independent of its upregulation, and another non-ICAM-1-dependent mechanism, in which carbohydrates expressed on HUVEC recognize L-selectin on PMN.
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PMID:Reactive oxygen species rapidly increase endothelial ICAM-1 ability to bind neutrophils without detectable upregulation. 751 10

We have demonstrated that the endothelial cell-derived superoxide anion is deeply involved in the endothelial cell injury induced by activated neutrophils (Fujita, H., Morita, I. and Murota, S. (1994) Arch. Biochem. Biophys. 309, 62-69). To clarify the mechanism underlying the increase in the endothelial cell-derived superoxide anion induced by activated neutrophils, the conversion of xanthine dehydrogenase (XD) to xanthine oxidase (XO) in cultured endothelial cells isolated from bovine carotid arteries was investigated. Although the endothelial cells expressed both XD and XO activity, the XO activity of unstimulated cells comprised about 12% of the total (XD + XO) activity. When endothelial cells were exposed to neutrophils activated with phorbol 12-myristate 13-acetate (PMA), XO activity rapidly increased about 3-fold over the control. Whereas treatment of endothelial cells with PMA alone or unstimulated neutrophils alone did not increase the XO activity at all. The increase in XO activity in endothelial cells was also observed on the treatment of the cells with neutrophils activated with leukotriene B4 or thrombin. To determine whether or not proteases released from activated neutrophils are involved in the increased conversion of XD to XO in endothelial cells, the effects of the elastase specific inhibitor, ONO-5046, and protease inhibitors, such as aprotinin, gabexate mesylate and urinastatin, were examined. However, these protease inhibitors did not suppress the conversion of XD to XO induced by PMA-activated neutrophils. Moreover, the treatment of endothelial cells with purified human neutrophil elastase and H2O2 also did not affect the conversion at all. In contrast, monoclonal antibodies against CD11a and CD18 significantly inhibited the increased conversion of XD to XO induced by PMA-activated neutrophils. Moreover, tyrosine kinase inhibitors such as staurosporin and herbimysine also inhibited the increased conversion of XD to XO induced by PMA-activated neutrophils. These results indicate that the adhesion of activated neutrophils to endothelial cells via CD11a/CD18-ICAM-1 is involved in the conversion of XD to XO in endothelial cells induced by activated neutrophils.
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PMID:Conversion of xanthine dehydrogenase to xanthine oxidase in bovine carotid artery endothelial cells induced by activated neutrophils: involvement of adhesion molecules. 769 38

Heat-shock protein 27 (HSP27) is a major target of phosphorylation upon cell stimulation with a variety of agents and has been suggested to have a phosphorylation-regulated function at the level of actin filaments. Here we investigated comparatively the mechanisms of HSP27 phosphorylation by oxidative stresses, exposures to tumor necrosis factor (TNF), heat shock and growth factors. Extracts of Chinese hamster or human cells exposed to H2O2, xanthine/xanthine oxidase, menadione or TNF contained up to 15-fold more HSP27 kinase activity than comparable extracts obtained from control cells. Induction of HSP27 kinase activity by TNF or H2O2 was completely inhibited by first treating the cells with the antioxidant N-acetyl-L-cysteine, suggesting that generation of reactive oxygen metabolites was the key triggering element of this induction. In contrast, prior treatment with acetylcysteine had no or little effect on the induction by thrombin, serum and heat shock. The kinase activity in extracts of cells stimulated by heat shock, H2O2, sodium arsenite, TNF or growth factors was identified by in-gel renaturation and purified approximately 8000-fold by sequential chromatography. In all cases, the induced kinase activity was entirely associated with two polypeptides of 45 kDa and 54 kDa, identified as mitogen-activated-protein kinase-activated protein (MAPKAP) kinase-2 based on its reactivation in vitro by 42/44-kDa MAP kinases, its antigenic properties and its substrate specificity. The 45/54-kDa HSP27 kinase may play an important role in the cell response to oxidative stress. Overexpression of the wild-type HSP27 but not of a nonphosphorylatable form of human HSP27 in Chinese hamster cells conferred resistance to actin fragmentation by oxidative stress generated by H2O2. It is concluded that activation of the 45/54-kDa HSP27 kinase is a common mechanism of HSP27 phosphorylation to which converge both oxyradical-dependent and oxyradical-independent pathways and which may participate in a homeostatic response to stress at the level of actin microfilament.
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PMID:Characterization of 45-kDa/54-kDa HSP27 kinase, a stress-sensitive kinase which may activate the phosphorylation-dependent protective function of mammalian 27-kDa heat-shock protein HSP27. 785 16

We have expressed, purified, and analyzed the iron-containing superoxide dismutase (FeSOD) of Escherichia coli with mutations directed at tyrosine position 34 to introduce phenylalanine (SODY34F), serine (SODY34S), or cysteine (SODY34C). FeSOD and mutant enzymes were purified from SOD-deficient cells using a GST-FeSOD fusion protein intermediate which was subsequently cleaved with thrombin and repurified. Specific activities were measured using the xanthine-xanthine oxidase method and gave 3148 u/mg for wild-type FeSOD. The SODY34S mutation virtually inactivates the enzyme (42 u/mg); mutation to cysteine greatly reduces activity (563 u/mg), but the SODY34F mutant retains nearly 40% of the activity of wild type (1205 u/mg). Fusion protein intermediates were also shown to be active and were demonstrated to protect SOD-deficient E. coli cells from the induced effects of oxidative stress, with growth rates directly proportional to the specific activities of the expressed mutant enzymes. SODY34F exhibited decreased thermal stability, reduced activity at high pH, and a pronounced increase in sensitivity to the inhibitor sodium azide compared with wild-type FeSOD. These results suggest that tyrosine at position 34 is multifunctional and plays a structural role (probably through hydrogen bonding to glutamine at position 69) in maintaining the integrity of the active site, a stabilizing role at high pH, and a steric role in obstructing access to the active site of both substrate and inhibitor molecules.
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PMID:The conserved residue tyrosine 34 is essential for maximal activity of iron-superoxide dismutase from Escherichia coli. 912 14

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