Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:1.17.3.2 (xanthine oxidase)
8,383 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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

Previously, our laboratory reported that lactosylceramide (LacCer) stimulated human aortic smooth muscle cell proliferation via specific activation of p44 mitogen-activated protein kinase (MAPK) in the p21(ras)/Raf-1/MEK2 pathway and induced expression of the transcription factor c-fos downstream to the p44 MAPK signaling cascade (Bhunia A. K., Han, H., Snowden, A., and Chatterjee S. (1996) J. Biol. Chem. 271, 10660-10666). In the present study, we explored the role of free oxygen radicals in LacCer-mediated induction of cell proliferation. Superoxide levels were measured by the lucigenin chemiluminescence method, MAPK activity was measured by immunocomplex kinase assays, and Western blot analysis and c-fos expression were measured by Northern blot assay. We found that LacCer (10 microM) stimulates endogenous superoxide production (7-fold compared with control) in human aortic smooth muscle cells specifically by activating membrane-associated NADPH oxidase, but not NADH or xanthine oxidase. This process was inhibited by an inhibitor of NADPH oxidase, diphenylene iodonium (DPI), and by antioxidants, N-acetyl-L-cysteine (NAC) or pyrrolidine dithiocarbamate. NAC and DPI both abrogated individual steps in the signaling pathway leading to cell proliferation. For example, the p21(ras).GTP loading, p44 MAPK activity, and induction of transcription factor c-fos all were inhibited by NAC and DPI as well as an antioxidant pyrrolidine dithiocarbamate or reduced glutathione (GSH). In contrast, depletion of GSH by L-buthionine (S, R)-sulfoximine up-regulated the above described signaling cascade. In sum, LacCer, by virtue of activating NADPH oxidase, produces superoxide (a redox stress signaling molecule), which mediates cell proliferation via activation of the kinase cascade. Our findings may explain the potential role of LacCer in the pathogenesis of atherosclerosis involving the proliferation of aortic smooth muscle cells.
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PMID:Redox-regulated signaling by lactosylceramide in the proliferation of human aortic smooth muscle cells. 918 53

Endothelial cells (ECs) are constantly exposed to blood pressure-induced mechanical strain. We have previously demonstrated that cyclic strain can induce gene expression of monocyte chemotactic protein-1 (MCP-1). The molecular mechanisms of gene induction by strain, however, remain unclear. Recent evidence indicates that intracellular reactive oxygen species (ROS) can act as a second messenger for signal transduction and thus affect gene expression. The potential role of ROS in strain-induced MCP-1 expression was investigated. ECs under cyclic strain induced a sustained elevated production of intracellular superoxide. ECs under strain or pretreated with either H2O2 or xanthine oxidase/hypoxanthine induced MCP-1 expression. Strain- or oxidant-induced MCP-1 mRNA levels could be inhibited by treating ECs with catalase or antioxidant N-acetyl-cysteine (NAC). Functional analysis of MCP-1 promoter and site-specific mutations indicates that the proximal tissue plasminogen activator-responsive element (TRE) in the -60-bp promoter region is sufficient for strain or H2O2 inducibility. Electrophoretic mobility shift assays demonstrated an increase of nuclear proteins binding to TRE sequences from ECs subsequent to strain or H2O2 treatment. NAC or catalase pretreatment of ECs inhibited the strain- or H2O2-induced AP-1 binding. These results clearly indicate that cyclic strain inducibility of MCP-1 in ECs uses the interaction of AP-1 proteins with TRE sites via the elevation of intracellular ROS levels in strained ECs. These findings emphasize the importance of intracellular ROS in the modulation of hemodynamic force-induced gene expression in vascular ECs.
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PMID:Cyclic strain-induced monocyte chemotactic protein-1 gene expression in endothelial cells involves reactive oxygen species activation of activator protein 1. 920 Oct 21

The effect of diethyldithiocarbamate (DDC) and sodium nitroprusside (SNP) on the killing of endothelial cells and on the release of arachidonate by mixtures of oxidants and membrane-damaging agents was studied in a tissue culture model employing bovine aortic endothelial cells labeled either with 51Chromium or 3arachidonic acid. While exposure to low, subtoxic concentrations of oxidants (reagent H2O2, glucose-oxidase generated peroxide, xanthine xanthine oxidase, AAPH-generated peroxyl radical, menadione-generated oxidants) did not result either in cell death or in the loss of membrane-associated arachidonic acid, the addition of subtoxic amounts of a variety of membrane-damaging agents (streptolysin S, PLA2, histone, taurocholate, wheatgerm agglutinin) resulted in a synergistic cell death. However, no significant amounts of arachidonate were released unless proteinases were also present. The addition to these reaction mixtures of subtoxic amounts of DDC (an SOD inhibitor and a copper chelator) not only very markedly enhanced cell death but also resulted in the release of large amounts of arachidonate (in the complete absence of added proteinases). Furthermore, the inclusion in DDC-containing reaction mixtures of subtoxic amounts of SNP, a generator of NO, further enhanced, in a synergistic manner, both cell killing and the release of arachidonate. Cell killing and the release of arachidonate induced by the DDC and SNP-containing mixtures of agonists were strongly inhibited by catalase, glutathione, N-acetyl cysteine, vitamin A, and by a nonpenetrating PLA2 inhibitor as well as by tetracyclines. A partial inhibition of cell killing was also obtained by 1,10-phenanthroline and by antimycin. It is suggested that DDC might amplify cell damage by forming intracellular, loosely-bound complexes with copper and probably also by depleting antioxidant thiols. It is also suggested that "cocktails" containing oxidants, membrane-damaging agents, DDC, and SNP might be beneficial for killing of tumor cells in vivo and for the assessment of the toxicity of xenobiotics in vitro.
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PMID:Diethyldithiocarbamate and nitric oxide synergize with oxidants and with membrane-damaging agents to injure mammalian cells. 935 Apr 19

When rat liver xanthine dehydrogenase was incubated with fluorodinitrobenzene (FDNB) at pH 8.5, the total enzyme activity decreased gradually to a limited value of initial activity with modification of two lysine residues in a similar way to the modification of bovine milk xanthine oxidase with FDNB (Nishino, T., Tsushima, K., Hille, R. and Massey, V. (1982) J. Biol. Chem. 257, 7348-7353). After modification with FDNB, the two peptides containing dinitrophenyl-lysine were isolated from the molybdopterin domain after proteolytic digestion and were identified as Lys754 and Lys771 by sequencing the peptides. During the modification of these lysine residues, xanthine dehydrogenase was found to be converted to an oxidase form in the early stage of incubation. Incorporation of the 3H-dinitrophenyl group into enzyme cysteine residues was 0.96 mol per enzyme FAD for 68% conversion to the oxidase form. The modified enzyme was reconverted to the dehydrogenase form by incubation with dithiothreitol with concomitant release of 3H-dinitrophenyl compounds. After modification with 3H-FDNB followed by carboxymethylation under denaturating conditions, the enzyme was digested with proteases. Three 3H-dinitrophenyl-labeled peptides were isolated and sequenced. The modified residues were identified to be Cys535, Cys992 and Cys1324. These residues are conserved among the all known mammalian enzymes, but Cys992 and Cys1324 are not conserved in the chicken enzyme. Cys1324 of the rat enzyme was found not to be involved in the conversion from the dehydrogenase to the oxidase by limited proteolysis experiments, but Cys535 and Cys992 which seemed to be modified alternatively with FDNB appear to be involved in the conversion.
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PMID:The conversion from the dehydrogenase type to the oxidase type of rat liver xanthine dehydrogenase by modification of cysteine residues with fluorodinitrobenzene. 936 59

Reactive oxygen species (ROS) have been suggested to act as cellular messengers that mediate signal transduction cascades in various cell types. However, little is known about their role in this capacity in the nervous system. We have begun to investigate the role of ROS, and that of nitric oxide (NO), in mediating mitogen-activated protein kinase (MAPK) signaling in rat hippocampal slices. Our studies have revealed that direct exposure of hippocampal slices to hydrogen peroxide, xanthine/xanthine oxidase (a superoxide-generating system), sodium nitroprusside (an NO donor compound), S-nitroso-N-acetylpenicillamine (an NO donor compound), or 3-morpholinosydnonimine (a compound that produces NO and superoxide) results in an enhancement in tyrosine phosphorylation of several proteins, including proteins with apparent molecular masses of 42 and 44 kDa. We investigated the possibility that these proteins correspond to the active forms of p42 MAPK and p44 MAPK. Hippocampal slices exposed to various ROS and NO donors resulted in increases in levels of the active forms of both p42 MAPK and p44 MAPK. The ROS- and NO-enhanced tyrosine phosphorylation and activation of p42 MAPK and p44 MAPK were inhibited by pretreatment with the antioxidant N-acetyl-L-cysteine. Our observations indicate that ROS and NO can mediate protein tyrosine phosphorylation and MAPK signaling in the hippocampus via a redox-sensitive mechanism and suggest a potential cellular mechanism for their effects in the nervous system.
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PMID:Stimulation of p42 and p44 mitogen-activated protein kinases by reactive oxygen species and nitric oxide in hippocampus. 948 20

S-Nitrosothiols (RSNO) occur in vivo and have been proposed as nitric oxide (.NO) storage and transport biomolecules. Still, the biochemical mechanisms by which RSNO release .NO in biological systems are not well defined, and in particular, the interactions between reactive oxygen species and RSNO have not been studied. In this work, we show that xanthine oxidase (XO), in the presence of purine (hypoxanthine, xanthine) or pteridine (lumazine) substrates, induces S-nitrosocysteine (CysNO) and S-nitrosoglutathione (GSNO) decomposition under aerobic conditions. The decomposition of RSNO by XO was inhibitable by copper-zinc superoxide dismutase, in agreement with the participation of superoxide anion (O-2) in the process. However, while superoxide dismutase could totally inhibit aerobic decomposition of GSNO, it was only partially inhibitory for CysNO. Competition experiments indicated that O-2 reacted with GSNO with a rate constant of 1 x 10(4) M-1.s-1 at pH 7.4 and 25 degreesC. The decomposition of RSNO was accompanied by peroxynitrite formation as assessed by the oxidation of dihydrorhodamine and of cytochrome c2+. The proposed mechanism involves the O-2-dependent reduction of RSNO to yield .NO, which in turn reacts fast with a second O-2 molecule to yield peroxynitrite. Under anaerobic conditions, CysNO incubated with xanthine plus XO resulted in CysNO decomposition, .NO detection, and cysteine and uric acid formation. We found that CysNO is an electron acceptor substrate for XO with a Km of 0.7 mM. In agreement with this concept, the enzymatic reduction of CysNO by XO was inhibitable by oxypurinol and diphenyliodonium, inhibitors that interfere with the catalytic cycle at the molybdenum and flavin sites, respectively. In conclusion, XO decomposes RSNO by O-2-dependent and -independent pathways, and in the presence of oxygen it leads to peroxynitrite formation.
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PMID:Xanthine oxidase-mediated decomposition of S-nitrosothiols. 952 75

1,1-Diphenyl-2-Picryl-Hydrazyl (DPPH), a stable free radical, has been used for detecting antioxidant activity in chemical analysis. However, it is still unknown if DPPH triggers free radical injury in cardiac tissue. In order to establish a simple free radical-injured isolated heart model, we investigated the action of DPPH on isolated guinea pig heart by Langendorff perfusion and compared it with cardiac effect of superoxide anion (O2.-), generated by the hypoxanthine (HX)-xanthine oxidase (XO) system. Free radical scavengers, dimethyl sulphoxide (DMSO), superoxide dismutase (SOD), and L-cysteine, were also used to analyze the characteristic of the DPPH free radical-derived cardiac dysfunction. In isolated guinea pig hearts, DPPH 100 nM and 250 nM in Krebs-Henseleit solution significantly decreased the left ventricular developed pressure (LVDP) and maximum velocity changes of left ventricular pressure (+/-LVdP/dtmax), elevated the left ventricular end-diastolic pressure (LVEDP), and increased lactate dehydrogenase (LDH) release and thiobarbituric acid-reactive substances (TBARS) formation in cardiac tissue. The cardiac dysfunction induced by DPPH 250 nM was more intense than 100 nM. L-Cysteine improved the DPPH-impaired cardiac function, while DMSO and SOD had no beneficial effect on this injury. The cardiac membrane fluidity was decreased by DPPH. Free radical signals, detected by electron spin resonance (ESR) in the DPPH-injured heart, were reduced by L-cysteine-treatment. These results suggest that DPPH free radical-induced cardiac dysfunction is attributed to neither the superoxide anion nor the hydroxyl radical. In conclusion, our data indicate that DPPH-induced isolated heart dysfunction serves as a simple and reproducible free radical-injured heart model.
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PMID:A simple reproducible model of free radical-injured isolated heart induced by 1,1-diphenyl-2-picryl-hydrazyl (DPPH). 969 63

In this study we have investigated some chemical properties and the biological role of thiazolidine compounds, obtained by condensation of aminothiols (L- or D-cysteine, cysteamine) with pyridoxal-5'-phosphate. These products have been tested in presence of rat liver extracts (supernatant and mitochondria); bacterial suspensions and enzymes (L- or D-aminoacid oxidase, xanthine oxidase) with interesting results which gives evidence to a biological role. Their formation in vivo may represent the regulation of intracellular levels of pyridoxal-5'-phosphate and aminothiols. Moreover, we have analysed the two diastereoisomers of the thiazolidine compounds derived from L-cysteine and D-cysteine: we have succeeded to distinguish by NMR analysis the cis and the trans forms, concluding that the interconversion of the free forms is extremely rapid at pH 7: thus, it may be relevant for the protein bound forms.
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PMID:Some chemical properties and biological role of thiazolidine compounds. 977 14

Time courses of total (GSH-t), disulfide (GSSG), and mixed disulfide (PSSG) forms of glutathione were studied in chicken blood submitted to oxidative stress induced by diamide or by the reactive oxygen species (ROS)-producing system xanthine/xanthine oxidase (X/XO). Diamide-treated blood induced an immediate increase in GSSG and PSSG, while X/XO produced a slow and sustained stress with increased values of GSSG and PSSG only after 30 and/or 60 min of incubation. Both total protein S-thiolation (mixed disulfide with glutathione) and dethiolation and hemoglobin A S-thiolation and dethiolation were clearly observed. Hemoglobin A (Hb A) was the major S-thiolated protein. We further characterized chicken Hb S-thiolation through the reaction of Hb with GSSG or the GSH/GSSG redox couple. Methemoglobin levels did not change with diamide or with X/XO treatment. Present results suggest that the most reactive cysteine pair of Hb A, the major chicken Hb, might function as an antioxidant under in vivo oxidative stress conditions.
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PMID:Oxidative stress causes intracellular reversible S-thiolation of chicken hemoglobin under diamide and xanthine oxidase treatment. 978 42


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