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)

1. The effects of inhibitors and activators on the azo- and nitro-reductases of Ascaris lumbricoides var suum have been investigated. Both types of reduction were inhibited by FAD, FMN, riboflavin, allopurinol, dicoumarol, 5-nitro-2-furaldehyde, azide and cyanide at concentrations of 1 mM. Neither reaction was inhibited by menadione, nitrofurantoin, SKF 525-A or fluoride. Both reactions were stimulated by addition of hypoxanthine. 2. The enzyme preparation contained no detectable aldehyde oxidase or xanthine oxidase activity. 3. The differences in the effects of flavins and inhibitors on mammalian and nematode azo- and nitro-reductases might have practical significance in the development of anthelmintic synergists.
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PMID:The effect of flavins and enzyme inhibitors on 4-nitrobenzoic acid reductase and azo reductase of Ascaris lumbricoides var suum. 5 46

1. The mid-point reduction potentials of the various groups in xanthine oxidase from bovine milk were determined by potentiometric titration with dithionite in the presence of dye mediators, removing samples for quantification of the reduced species by e.p.r. (electron-paramagnetic-resonance) spectroscopy. The values obtained for the functional enzyme in pyrophosphate buffer, pH8.2, are: Fe/S centre I, -343 +/- 15mV; Fe/S II, -303 +/- 15mV; FAD/FADH-; -351 +/- 20mV; FADH/FADH2, -236 +/-mV; Mo(VI)/Mo(V) (Rapid), -355 +/- 20mV; Mo(V) (Rapid)/Mo(IV), -355 +/- 20mV. 2. Behaviour of the functional enzyme is essentially ideal in Tris but less so in pyrophosphate. In Tris, the potential for Mo(VI)/Mo(V) (Rapid) is lowered relative to that in pyrophosphate, but the potential for Fe/S II is raised. The influence of buffer on the potentials was investigated by partial-reduction experiments with six other buffers. 3. Conversion of the enzyme with cyanide into the non-functional form, which gives the Slow molybdenum signal, or alkylation of FAD, has little effect on the mid-point potentials of the other centres. The potentials associated with the Slow signal are: Mo(VI)/Mo(V) (Slow), -440 +/- 25mV; Mo(V) (Slow)/Mo(IV), -480 +/- 25 mV. This signal exhibits very sluggish equilibration with the mediator system. 4. The deviations from ideal behaviour are discussed in terms of possible binding of buffer ions or anti-co-operative interactions amongst the redox centres.
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PMID:Oxidation-reduction potentials of molybdenum, flavin and iron-sulphur centres in milk xanthine oxidase. 18 52

Redox potentials for the various centres in the enzyme xanthine dehydrogenase (EC 1.2.1.37) from turkey liver determined by potentiometric titration in the presence of mediator dyes, with low-temperature electron-paramagnetic-resonance spectroscopy. Values at 25 degrees C in pyrophosphate buffer, pH 8.2, are: Mo(VI)/Mo(V)(Rapid),-350 +/- 20mV; Mo(V) (Rapid)/Mo(IV), -362 +/- 20mV; Fe-S Iox./Fe-S Ired., -295 +/- 15mV; Fe-S IIox./Fe-S IIred., -292 +/- 15mV; FAD/FADH,-359+-20mV; FADH/FADH2, -366 +/- 20mV. This value of the FADH/FADH2 potential, which is 130mV lower than the corresponding one for milk xanthine oxidase [Cammack, Barber & Bray (1976) Biochem. J. 157, 469-478], accounts for many of the differences between the two enzymes. When allowance is made for some interference by desulpho enzyme, then differences in the enzymes' behaviour in titration with xanthine [Barber, Bray, Lowe & Coughlan (1976) Biochem. J. 153, 297-307] are accounted for by the potentials. Increases in the molybdenum potentials of the enzymes caused by the binding of uric acid are discussed. Though the potential of uric acid/xanthine (-440mV) is favourable for full reduction of the dehydrogenase, nevertheless, during turnover, for kinetic reasons, only FADH and very little FADH2 is produced from it. Since only FADH2 is expected to react with O2, lack of oxidase activity by the dehydrogenase is explained. Reactivity of the two enzymes with NAD+ as electron acceptor is discussed in relation to the potentials.
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PMID:Oxidation--reduction potentials of turkey liver xanthine dehydrogenase and the origins of oxidase and dehydrogenase behaviour in molybdenum-containing hydroxylases. 86 27

Bovine milk xanthine oxidase (xanthine:oxygen oxidoreductase, EC 1.2.3.2) has been purified by a modified method without the use of proteases, and its structure has been analyzed by polyacrylamide gel electrophoresis. Native xanthine oxidase is found to consist of only two polypeptide chains A with molecular weights of 150 000 each. These chains have NH2-terminal methionine. Limited proteolysis with trypsin, chymotrypsin, or subtilisin at pH 8 did not affect molecular weight and activities of the enzyme while each of the A chains was cleaved under these conditions to three fragments C, E, and F with molecular weights of 92 00, 42 000 and 20 000, respectively. These fragments remained bound to each other and were relatively resistant to subsequent proteolysis. The isolation of xanthine oxidase in the presence of pancreatin as described by Hart et al. (1970, Biochem. J. 116, 851) gives partially digested enzyme composed mainly of chains C, E (Mr 35 000) and a small component (Mr approx. 15 0-0). The action of subtilisin on xanthine oxidase at pH 11 resulted in complete digestion of E chains, FAD separation, and total loss of xanthine:oxygen oxidoreductase activity while xanthine:indophenol oxidoreductase activity was relatively little affected. The residual enzyme has a molecular weight of about 200 000, is composed mainly of two C chains (and may probably contain F and/or proteolytic fragments of low molecular weight), contains molybdenum, and does not contain FAD.
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PMID:Subunit structure of bovine milk xanthine oxidase. Effect of limited cleavage by proteolytic enzymes on activity and structure. 126 10

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

Milk xanthine oxidase (XO) has been prepared in a dehydrogenase form (XDH) by purifying the enzyme in the presence of 2.5 mM dithiothreitol. Unlike XO, which reacts rapidly only with oxygen and not with NAD, the XDH form of the enzyme reacts rapidly with NAD. XDH has a turnover number for the NAD-dependent conversion of xanthine to urate of 380 mol/min/mol at pH 7.5, 25 degrees C, with a Km = < or = 1 microM for xanthine and a Km = 7 microM for NAD, but has very little O2-dependent activity. There is evidence that the two forms of the enzyme have different flavin environments: XDH stabilizes the neutral form of the flavin semiquinone and XO does not. Further, XDH binds the artificial flavin 8-mercapto-FAD in its neutral form, shifting the pK of this flavin by 5 pH units, while XO binds 8-mercapto-FAD in its benzoquinoid anionic form. XDH can be converted back to the XO form by the addition of three to four equivalents of the disulfide-forming reagent 4,4'-dithiodipyridine, suggesting that, in the XDH form of the enzyme, disulfide bonds are broken; this may cause a conformational change which creates a binding site for NAD and changes the protein structure near the flavin.
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PMID:Purification and properties of milk xanthine dehydrogenase. 132 33

The rates of NADH oxidation in presence of xanthine oxidase increase to a small and variable extent on addition of high concentrations of lactate dehydrogenase and other dehydrogenases. This heat stable activity is similar to polyvanadate-stimulation with respect to pH profile and SOD sensitivity. Isocitric dehydrogenase (NADP-specific) showed heat labile, SOD-sensitive polyvanadate-stimulated NADH oxidation activity. Polyvanadate-stimulated SOD-sensitive NADH oxidation was also found to occur with riboflavin, FMN and FAD in presence of a non-specific protein, BSA, suggesting that some flavoproteins may possess this activity.
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PMID:Stimulation of NADH oxidation by xanthine oxidase and polyvanadate in presence of some dehydrogenases and flavin compounds. 178 72

31P ENDOR spectra are described for three different molybdenum(V) species in reduced xanthine oxidase samples. The spectra were not affected by removing the FAD from the enzyme, implying that this is located at some distance from molybdenum. Furthermore, in confirmation of the work of J. L. Johnson, R. E. London, and K. V. Rajagopalan [(1989) Proc. Natl. Acad. Sci. U.S.A. 86, 6493-6497], NMR and chemical analysis of the phosphate content of highly purified xanthine oxidase showed there are only three phosphate residues per subunit of the enzyme. It is concluded that the ENDOR features are due to hyperfine coupling of the phosphate group of the pterin cofactor to the molybdenum atom. Evaluation of the dipolar component of the coupling has permitted estimation of the molybdenum-phosphorus distances as 7-12 A. This implies that the cofactor is in an extended conformation in the enzyme molecule. Less detailed 31P ENDOR data on sulfite oxidase are consistent with a similar conformation for the cofactor in this enzyme.
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PMID:31P ENDOR studies of xanthine oxidase: coupling of phosphorus of the pterin cofactor to molybdenum (V). 185 Feb 96

The steady-state and rapid kinetic properties of xanthine oxidase containing a series of FAD analogs of varying reduction potential have been investigated. From steady-state analysis, Vmax is found to exhibit a sigmoidal dependence on the flavin midpoint potential in the homologous series. This dependence is accurately described by a model in which the rate of catalysis is attenuated by the amount of partially reduced enzyme generated during turnover possessing an unfavorable distribution of reducing equivalents among the several redox-active centers of the protein. The model assumes that reducing equivalents equilibrate among these centers rapidly compared to the limiting rates for the reductive and oxidative half-reactions. This assumption is borne out by a quantitative analysis of the reductive and oxidative half-reactions of the several enzyme forms investigated in detail. It is demonstrated in these studies that xanthine oxidase containing low potential flavin derivatives such as 1-deaza, 6-hydroxy, or 8-hydroxy FAD exhibits low turnover not because of inherently slow rates of reduction by xanthine or oxidation by molecular oxygen, but because in partially reduced enzyme generated in the course of turnover reducing equivalents are distributed within the enzyme in such a way that the enzyme can participate in neither the reductive nor oxidative half-reactions. These results provide confirmation of the operation of a thermodynamic control mechanism in a simple electron-transferring system.
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PMID:The kinetic behavior of xanthine oxidase containing chemically modified flavins. 189 27

Electron transfer within milk xanthine oxidase has been examined by the technique of pulse radiolysis. Radiolytically generated N-methylnicotinamide radical or 5-deazalumiflavin radical has been used to rapidly and selectively introduce reducing equivalents into the enzyme so that subsequent equilibration among the four redox-active centers of the enzyme (a molybdenum center, two iron-sulfur centers, and FAD) could be monitored spectrophotometrically. Experiments have been performed at pH 6 and 8.5, and a comprehensive scheme describing electron equilibration within the enzyme at both pH values has been developed. All rate constants ascribed to equilibration between specific pairs of centers in the enzyme are found to be rapid relative to enzyme turnover under the same conditions. Electron equilibration between the molybdenum center and one of the iron-sulfur centers of the enzyme (tentatively assigned Fe/S I) is particularly rapid, with a pH-independent first-order rate constant of approximately 8.5 x 10(3) s-1. The results unambiguously demonstrate the role of the iron-sulfur centers of xanthine oxidase in mediating electron transfer between the molybdenum and flavin centers of the enzyme.
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PMID:Electron transfer in milk xanthine oxidase as studied by pulse radiolysis. 200


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