Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P47989 (xanthine oxidase)
 8,633 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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.
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PMID:Regulation, purification, and properties of xanthine dehydrogenase in Neurospora crassa. 14 74

Two proteins (P1 and P2, with weights of 57,500 and 27,500 respectively) were isolated from Euglena gracilis. Both proteins show cyanide-insensitive superoxide dismutase activity in the "classical" superoxide dismutase assay, using xanthine-xanthine oxidase as O2.- generator. If O2.- is generated chemically (autoxidation of reduced anthraquinone), photochemically (illuminated riboflavine) or pulse radiolytically, only protein P1 but not P2 shows SOD activity. Protein P1 contains 1 g atom (determined: 0.82) iron (no Mn or Cu) per mole protein and may thus be defined as iron-superoxide dismutase. Protein P2, showing the spectral properties of a flavoprotein, exhibits the activities of ferredoxin-NADP-oxidoreductase and "diaphorase". The cyanide-insensitive SOD-activity of this Diaphorase" in the xanthine oxidase-assay for superoxide dismutase makes this classical and commonly used test unreliable for assay cyanide insensitive SOD activities. The existence of the "prokaryote-type" of superoxide dismutase (Fe-SOD) in Euglena gracilis is exceptional for an eukaryotic, autotrophically grown organisms.
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PMID:Cyanide insensitive iron superoxide dismutase in Euglena gracilis. Comparison of the reliabilities of different test systems for superoxide dismutases. 22 43

Xanthine dehydrogenase has been purified to homogeneity by conventional procedures from the wild-type strain of the fruit fly Drosophila melanogaster, as well as from a rosy mutant strain (E89----K, ry5231) known to carry a point mutation in the iron-sulfur domain of the enzyme. The wild-type enzyme had all the specific properties that are peculiar to the molybdenum-containing hydroxylases. It had normal contents of molybdenum, the pterin molybdenum cofactor, FAD, and iron-sulfur centers. EPR studies showed its molybdenum center to be quite indistinguishable from that of milk xanthine oxidase. As isolated, only about 10% of the enzyme was present in the functional form, with most or all of the remainder as the inactive desulfo form. It is suggested that this may be present in vivo. Extensive proteolysis accompanied by the development of oxidase activity took place during isolation, but dehydrogenase activity was retained. EPR properties of the reduced iron-sulfur centers, Fe-SI and Fe-SII, in the enzyme are very similar to those of the corresponding centers in milk xanthine oxidase. The E89----K mutant enzyme variant was in all respects closely similar to the wild-type enzyme, with the exception that it lacked both of the iron-sulfur centers. This was established both by its having the absorption spectrum of a simple flavoprotein and by the complete absence of EPR signals characteristic of iron-sulfur centers in the reduced enzyme. Despite the lack of iron-sulfur centers, the mutant enzyme had xanthine:NAD+ oxidoreductase activity indistinguishable from that of the wild-type enzyme. Stopped-flow measurements indicated that, as for the wild-type enzyme, reduction of the mutant enzyme was rate-limiting in turnover. Thus, the iron-sulfur centers appear irrelevant to the normal turnover of the wild-type enzyme with these substrates. However, activity to certain oxidizing substrates, particularly phenazine methosulfate, is abolished in the mutant enzyme variant. This is one of the first examples of deletion by genetic means of iron-sulfur centers from an iron-sulfur protein. The relevance of our findings both to the roles of iron-sulfur centers in other systems and to the nature of the oxidizing substrate for the Drosophila enzyme in vivo are briefly discussed.
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PMID:Xanthine dehydrogenase from Drosophila melanogaster: purification and properties of the wild-type enzyme and of a variant lacking iron-sulfur centers. 131 86

Diphenylene iodonium (Ph2I), a lipophilic reagent, is an efficient inhibitor of the production of O2- by the activated NADPH oxidase of bovine neutrophils. In a cell-free system of NADPH oxidase activation consisting of neutrophil membranes and cytosol from resting cells, supplemented with guanosine 5'-[gamma-thio]triphosphate, MgCl2 and arachidonic acid, or in membranes isolated from neutrophils activated by 4 beta-phorbol 12-myristate 13-acetate, addition of a reducing agent, e.g. NADPH or sodium dithionite, markedly enhanced inhibition of the NADPH oxidase by Ph2I. The membrane fraction was found to contain the Ph2I-sensitive component(s). In the presence of a concentration of Ph2I sufficient to fully inhibit O2- production (around 10 nmol/mg membrane protein), addition of catalytic amounts of the redox mediator dichloroindophenol (Cl2Ind) resulted in a by-pass of the electron flow to cytochrome c, the rate of which was about half of that determined in non-inhibited oxidase. A marked increase in the efficiency of this by-pass was achieved by addition of sodium deoxycholate. The Cl2-Ind-mediated cytochrome c reduction was negligible in membranes isolated from resting neutrophils. At a higher concentration of Ph2I (100 nmol/mg membrane protein), the Cl2Ind-mediated cytochrome c reductase activity was only half inhibited, which indicated that, in the NADPH oxidase complex, there are at least two Ph2I sensitive components, differing by their sensitivity to the inhibitor. At low concentrations of Ph2I (less than 10 nmol/mg protein), the spectrum of reduced cytochrome b558 in isolated neutrophil membranes was modified, suggesting that the component sensitive to low concentrations of Ph2I is the heme binding component of cytochrome b558. Higher concentrations of Ph2I were found to inhibit the isolated NADPH dehydrogenase component of the oxidase complex. A number of membrane and cytosolic proteins were labeled by [125I]Ph2I. However, the radiolabeling of a membrane-bound 24-kDa protein, which might be the small subunit of cytochrome b558, responded more specifically to the conditions of activation and reduction which are required for inhibition of O2- production by Ph2I. The O2(-)-generating form of xanthine oxidase was also inhibited by Ph2I. Inhibition of xanthine oxidase, a non-heme iron flavoprotein, by Ph2I had a number of features in common with that of the neutrophil NADPH oxidase, namely the requirement of reducing conditions for inhibition of O2- production by Ph2I and the induction of a by-pass of electron flow to cytochrome c by Cl2Ind in the inhibited enzyme, suggesting some similarity in the molecular organization of the two enzymes.
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PMID:Diphenylene iodonium as an inhibitor of the NADPH oxidase complex of bovine neutrophils. Factors controlling the inhibitory potency of diphenylene iodonium in a cell-free system of oxidase activation. 132 36

Xanthine oxidase (XO), a molybdo-flavoprotein enzyme involved in purine degradation, was localized immunocytochemically in rat hepatocytes by high-resolution immunoelectron microscopy. XO was isolated from rat liver and a 150 KD polypeptide was purified. Antibodies were raised in rabbits. Small pieces of fresh liver were quickly frozen by contact with a copper block pre-cooled with liquid helium and were freeze-substituted with either 2.5% OsO4 or 0.2% glutaraldehyde in acetone. They were then warmed and embedded in Epon-Araldite or Araldite 6005. Resin sections were treated by indirect immunostaining using anti-rat liver XO antibody and protein A-gold. The labeling pattern was clearly over the cytosol and not on cell organelles. A few gold particles were found over the mitochondrial matrix, but not over the endoplasmic reticulum, Golgi apparatus, lysosomes, or peroxisomes, including their crystalloid core. These results are consistent with those of the biochemical assay of XO in this study. The significance of the occasional immunolabeling of the mitochondrial matrix remains obscure, since biochemical determinations in this study indicate no XO activity in the mitochondrial fraction.
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PMID:Subcellular localization of xanthine oxidase in rat hepatocytes: high-resolution immunoelectron microscopic study combined with biochemical analysis. 161 76

Few nonphagocytic cells are known to generate reactive oxygen intermediates. Based on horseradish peroxidase-dependent, catalase-inhibitable oxidation of fluorescent scopoletin, seven human tumor cell lines constitutively elaborated H2O2 at rates (up to 0.5 nmol/10(4) cells/h) large enough that cumulative amounts at 4 h were comparable to the amount of H2O2 produced by phorbol ester-triggered neutrophils. Superoxide dismutase-inhibitable ferricytochrome c reduction was detectable at much lower rates. H2O2 production was inhibited by diphenyleneiodonium, a flavoprotein binder (concentration producing 50% inhibition, 0.3 microM), and diethyldithiocarbamate, a divalent cation chelator (concentration producing 50% inhibition, 3 microM), but not by cyanide or azide, inhibitors of electron transport, or by agents that inhibit xanthine oxidase, polyamine oxidase, or cytochrome P450. Cytochrome b559, present in human phagocytes and lymphocytes, was undetectable in these tumor cells by a sensitive spectrophotometric method. Mouse fibroblasts transfected with human tyrosinase complementary DNA made melanin, but not H2O2. Constitutive generation of large amounts of reactive oxygen intermediates, if it occurs in vivo, might contribute to the ability of some tumors to mutate, inhibit antiproteases, injure local tissues, and therefore promote tumor heterogeneity, invasion, and metastasis.
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PMID:Production of large amounts of hydrogen peroxide by human tumor cells. 184 17

The flavoprotein nitroreductases NADPH:cytochrome P-450 reductase and xanthine oxidase catalyzed the cofactor-dependent anaerobic nitro group reduction and covalent binding to protein sulfhydryl groups of the 5-nitroimidazole substrate ronidazole [1-methyl-5-nitroimidazole-2-yl)-methyl carbamate). Studies with variously radiolabeled ronidazole molecules demonstrated that the imidazole ring was intact while greater than 80% of the C-4 3H and 2-carbamoyl group were lost from the covalently bound product. The stoichiometry of cofactor consumption during the enzyme-catalyzed reduction of the substrate could not be determined, so a model nitroreductase system which utilized dithionite as the reductant and agarose-immobilized cysteine as the target for alkylation was developed. Two moles of dithionite was consumed per mole of substrate for maximal reduction of uv absorbance due to the nitro group, for maximal release of C-4 3H, and for maximal covalent binding to agarose-immobilized cysteine. These results indicate that four electrons are required for the reductive activation of the substrate, consistent with formation of a hydroxylamine reactive intermediate. Covalent binding of variously radiolabeled substrate molecules after dithionite reduction exhibited the same labeling pattern as flavoprotein-catalyzed covalent binding, suggesting that covalent binding is mediated by the same species in both chemical and biological systems. The data are consistent with a mechanism where the substrate undergoes four-electron reduction to form a hydroxylamine, which is susceptible to nucleophilic attack at C-4. When water attacks C-4, the 2-carbamoyl group can eliminate to form a Michael-like acceptor which adds thiols at the 2-methylene position.
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PMID:Mechanism of reductive activation of a 5-nitroimidazole by flavoproteins: model studies with dithionite. 312 79

Xanthine oxidase is a flavoprotein which directly catalyses the oxidation of xanthine and hypoxanthine by oxygen or by potassium ferricyanide as an artificial acceptor of protons. In doing so, the potassium ferricyanide is reduced into potassium ferrocyanide which in the presence of manganese(II)ions leads to the manganese(II)ferrocyanide which is insoluble in water and in organic solvents. The latter is deposited on the areas with enzyme activity and marks them under the electron microscope. After the detection of the xanthine oxidase in rat liver on ultrathin non-contrasted sections, it was observed that the fine granular reaction product was deposited only on the peroxisomes of the hepatocytes. A greater quantity of the reaction product is deposited on the outer membrane and the matrix and a smaller one on the nucleoid of these cell organelles. No deposition of the reaction product was observed on the other cell structures. The method can be used for the study of purine metabolism on the cellular level as well as for the specific ultracytochemical detection of the peroxisomes.
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PMID:[Ultracytochemical demonstration of enzymes by reduction of potassium hexacyanoferrate (III). I. A method for demonstration of xanthine oxidase]. 313 15

Lodoxamide tromethamine and several other anti-allergy drugs, i.e. inhibitors of rat passive cutaneous anaphylaxis, are inhibitors of purified xanthine oxidase. Inhibition is noncompetitive with Ki's in the 1-13 micromolar range. Lodoxamide tromethamine had no effect on another flavoprotein, glucose oxidase. Other studies have shown several of these drugs are inhibitors of aldose reductase. It is speculated that the anti-allergy drugs inhibit mediator release from mast cells by blocking univalent electron transfers which are essential for release.
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PMID:Inhibition of oxidative enzymes by anti-allergy drugs. 612 76

Carbon monoxide:methylene blue oxidoreductase, the key enzyme of CO-oxidation in energy metabolism of the carboxydobacterium Pseudomonas carboxydovorans, has been isolated in good yield and purity and found to contain FAD, molybdenum, iron, and labile sulfide in the ratio of 1:1:4:4. The enzyme is, therefore, a new molybdenum-containing iron-sulfur flavoprotein, exhibiting chemical and spectral properties quite similar to those of xanthine oxidase. Analytical data on the spectral characteristics of the enzyme in the oxidized and various reduced states are presented. Carbon monoxide:methylene blue oxidoreductase turned out to be photoreducible in the presence of EDTA and urea and was subject to reoxidation by air oxygen; no flavoprotein semiquinone was formed. Unphysiological electron acceptors, e.g. methylene blue, were used as oxidizing substrates whereas NAD or NADP turned out to be ineffective. Methylene blue reduction with CO was not affected by the presence of allopurinol, and carbon monoxide:methylene blue oxidoreductase was not able to catalyze the reduction of methylene blue with xanthine, adenine, or aldehydes. CO was the only reducing substrate used by the enzyme. Carbon monoxide:methylene blue oxidoreductase formed no sulfite adduct, and the reactivity with ferricyanide or cytochrome c was significant but slow. As known for other molybdenum hydroxylases, carbon monoxide:methylene blue oxidoreductase was rapidly inactivated by methanol, but the enzyme exhibited no ability to catalyze the oxidation of NADH with methylene blue, and NAD was not able to overcome methanol inhibition.
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PMID:Chemical and spectral properties of carbon monoxide: methylene blue oxidoreductase. The molybdenum-containing iron-sulfur flavoprotein from Pseudomonas carboxydovorans. 627 81


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