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Turkey liver xanthine dehydrogenase. Reactivation of the cyanide-inactivated enxyme by sulphide and by selenide.

ABSTRACT: 1. Turkey liver xanthine dehydrogenase engaged in catalysing the oxidation of xanthine by dichlorophenol-indophenol was progressively inactivated by methanol. This inactivation was reversible by NAD(+). 2. Reaction with arsenite and with cyanide, in each case first-order with respect to enzyme, resulted in characteristic alterations in the visible absorption spectrum of the enzyme. The rate of spectral change on reaction with either agent paralleled the rate of loss of enzyme activity. 3. Cyanide inactivation was accompanied by elimination from the enzyme of sulphur as thiocyanate. Partial restoration of activity was effected by incubation with sulphide or with selenide. The results suggest that turkey liver xanthine dehydrogenase, like milk xanthine oxidase (Massey & Edmonson, 1970), contains at the active centre a cyanolysable persulphide group essential to catalytic activity and that selenium may replace sulphur in this group to give an active enzyme. 4. Incubation of the native enzyme with sulphide or with selenide resulted in the rapid loss of half of the xanthine-oxidizing activity, apparently by disrupting the molybdenum and (Fe/S)II loci. This may indicate non-equivalence of the intramolecular electron-transfer systems.

SUBMITTER: Cleere WF

PROVIDER: S-EPMC1168388 | biostudies-other | 1974 Nov

REPOSITORIES: biostudies-other

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Stopped-flow spectrophotometric studies on the reaction of turkey liver xanthine dehydrogenase with reducing substrates.

Project description:The kinetics of reduction of turkey liver xanthine dehydrogenase by substrates were investigated by stopped-flow spectrophotometry. The results may be explained in terms of the known redox potentials of the various centres in the enzyme [Barber, Bray, Cammack & Coughlan (1977) Biochem. J. 163, 279-289]. They are, morover, consistent with the scheme [Olson, Ballou, Palmer & Massey (1974) J. Biol. Chem. 249, 4363-4382] in which reduction occurs in three consecutive steps, one molecule of substrate reacting with the active site at each step. First-order rate constants believed to correspond respectively to the combined first and second steps and to the third step in the reduction by excess of xanthine and of NADH were determined. The rates of reaction with these substrates in the combined first and second steps are independent of the degree of enzyme functionality.

| S-EPMC1184136 | biostudies-other

Turkey liver xanthine dehydrogenase: properties of the enzyme dependent on the content of functional active sites.

Project description:Turkey liver xanthine dehydrogenase containing the full complement of molybdenum, flavin and iron-sulphur prosthetic groups is, as normally isolated, a mixture of functional and non-functional enzyme. The latter apparently lacks the cyanolysable persulphide groups essential to the oxidation of xanthine and to interaction with arsenite. These groups are not required for the oxidation of NADH by Methylene Blue. That KI treatment effects a differential release of flavin from xanthine-prereduced and NADH-prereduced enzyme merely reflects the degree of functionality of the preparations used and may not be taken as evidence for non-equivalence of the flavin chromophores.

| S-EPMC1168404 | biostudies-other

Oxidation--reduction potentials of turkey liver xanthine dehydrogenase and the origins of oxidase and dehydrogenase behaviour in molybdenum-containing hydroxylases.

Project description: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.

| S-EPMC1164694 | biostudies-other

Turkey liver xanthine dehydrogenase. Relation between nicotinamide-adenine dinucleotide oxidoreductase activity and the content of functional enzyme.

Project description:The turkey liver xanthine dehydrogenase-catalysed oxidation of NADH by Methylene Blue, by ferricyanide or by O2 is not dependent on the integrity of the active-centre persulphide groups. By contrast, the NADH-dichlorophenol-indophenol oxidoreductase and NADH-trinitrobenzenesulphonate oxidoreductase activities are directly proportional to the content of functional enzyme. These findings help to identify the sites of egress of electrons to oxidizing substrates.

| S-EPMC1163846 | biostudies-other

Studies by electron-paramagnetic-resonance spectroscopy and stopped-flow spectrophotometry on the mechanism of action of turkey liver xanthine dehydrogenase.

Project description:Studies by e.p.r. (electron-paramagnetic-resonance) spectroscopy and by stopped-flow spectrophotometry on turkey liver xanthine dehydrogenase revealed strong similarities to as well as important differences from the Veillonella alcalescens xanthine dehydrogenase and milk xanthine oxidase. The turkey enzyme is contaminated by up to three non-functional forms, giving molybdenum e.p.r. signals designated Resting I, Resting II and Slow. Slow and to a lesser extent Resting I signals are like those from the Veillonella enzyme, whereas Resting II is very like a resting signal described by K. V. Rajagopolan, P. Handler, G. Palmer & H. Beinert (1968) (J. Biol. Chem. 243, 3784-3796) for aldehyde oxidase. Another non-functional form that gives the Inhibited signal is produced on treatment of the enzyme with formaldehyde. Stopped-flow measurements at 450 nm show that, as for the milk enzyme, reduction by xanthine is rate-limiting in enzyme turnover. The active enzyme gives rise to Very Rapid and Rapid molybdenum(V) e.p.r. signals, as well as to an FADH signal. That these signals are almost indistinguishable from those of the milk enzyme, confirms the similarities between the active sites. There are two types of iron-sulphur centres that give signals like those in the milk enzyme, though with slightly different parameters. Quantitative reduction titration of the functional enzyme with xanthine revealed two important differences between the turkey and the milk enzymes. First, the turkey enzyme FADH/FADH2 system has a redox potential sufficiently low that xanthine is incapable of reducing the flavin completely. This finding presumably explains the very low oxidase activity. Secondly, whereas the Fe/S II chromophore in the milk enzyme has a relatively high redox potential, for the turkey enzyme the value of this potential is lower and similar to that of its Fe/S I chromophore.

| S-EPMC1172575 | biostudies-other

Kinetic mechanism of chicken liver xanthine dehydrogenase.

Project description:The kinetic behaviour of chicken-liver xanthine dehydrogenase (xanthine/NAD+ oxidoreductase; EC 1.2.1.37) has been studied. Steady-state results, obtained from a wide range of concentrations of substrates and products, were fitted by rational functions of degree 1:1, 1:2, 2:2 and 3:3 with respect to substrates, and 0:1, 1:1, 0:2 and 1:2 with regard to products, using a non-linear regression program which guarantees the fit. The goodness of fit was improved using a computer program that combines model discrimination, parameter refinement and sequential experimental design. The AIC and F tests were also used for model discrimination. For comparative purposes, the xanthine/oxygen oxidoreductase reaction was also studied. From the functions which give the maximum improvement, the complete rate equation was deduced. The significance of the terms was stated by the above methods. It was concluded that xanthine dehydrogenase requires a minimum mechanism of degree 1:1 for xanthine, 2:2 for NAD+, 1:1 for uric acid and 1:2 for NADH in the xanthine/NAD+ oxidoreductase reaction. These are the minimum degrees required but a rate equation of higher degree is not excluded.

| S-EPMC1148681 | biostudies-other

In vitro oxidative metabolism of 6-mercaptopurine in human liver: insights into the role of the molybdoflavoenzymes aldehyde oxidase, xanthine oxidase, and xanthine dehydrogenase.

Project description:Anticancer agent 6-mercaptopurine (6MP) has been in use since 1953 for the treatment of childhood acute lymphoblastic leukemia (ALL) and inflammatory bowel disease. Despite being available for 60 years, several aspects of 6MP drug metabolism and pharmacokinetics in humans are unknown. Molybdoflavoenzymes such as aldehyde oxidase (AO) and xanthine oxidase (XO) have previously been implicated in the metabolism of this drug. In this study, we investigated the in vitro metabolism of 6MP to 6-thiouric acid (6TUA) in pooled human liver cytosol. We discovered that 6MP is metabolized to 6TUA through sequential metabolism via the 6-thioxanthine (6TX) intermediate. The role of human AO and XO in the metabolism of 6MP was established using the specific inhibitors raloxifene and febuxostat. Both AO and XO were involved in the metabolism of the 6TX intermediate, whereas only XO was responsible for the conversion of 6TX to 6TUA. These findings were further confirmed using purified human AO and Escherichia coli lysate containing expressed recombinant human XO. Xanthine dehydrogenase (XDH), which belongs to the family of xanthine oxidoreductases and preferentially reduces nicotinamide adenine dinucleotide (NAD(+)), was shown to contribute to the overall production of the 6TX intermediate as well as the final product 6TUA in the presence of NAD(+) in human liver cytosol. In conclusion, we present evidence that three enzymes, AO, XO, and XDH, contribute to the production of 6TX intermediate, whereas only XO and XDH are involved in the conversion of 6TX to 6TUA in pooled HLC.

| S-EPMC4109211 | biostudies-literature

Crystal structures of bovine milk xanthine dehydrogenase and xanthine oxidase: structure-based mechanism of conversion.

Project description:Mammalian xanthine oxidoreductases, which catalyze the last two steps in the formation of urate, are synthesized as the dehydrogenase form xanthine dehydrogenase (XDH) but can be readily converted to the oxidase form xanthine oxidase (XO) by oxidation of sulfhydryl residues or by proteolysis. Here, we present the crystal structure of the dimeric (M(r), 290,000) bovine milk XDH at 2.1-A resolution and XO at 2.5-A resolution and describe the major changes that occur on the proteolytic transformation of XDH to the XO form. Each molecule is composed of an N-terminal 20-kDa domain containing two iron sulfur centers, a central 40-kDa flavin adenine dinucleotide domain, and a C-terminal 85-kDa molybdopterin-binding domain with the four redox centers aligned in an almost linear fashion. Cleavage of surface-exposed loops of XDH causes major structural rearrangement of another loop close to the flavin ring (Gln 423Lys 433). This movement partially blocks access of the NAD substrate to the flavin adenine dinucleotide cofactor and changes the electrostatic environment of the active site, reflecting the switch of substrate specificity observed for the two forms of this enzyme.

| S-EPMC27090 | biostudies-literature

Intermediate dehydrogenase-oxidase form of xanthine oxidoreductase in rat liver.

Project description:A spectrophotometric method for the determination of three forms of xanthine oxidoreductase, namely dehydrogenase (D), dehydrogenase-oxidase (D/O) and oxidase (O), is described. Enzymic fractions obtained from rat liver were found to contain either all three forms, or (under special conditions of preparation) only two forms, D and D/O. The conversion of form D leads to form D/O leads to form O in the presence of Cu2+ ions was shown. Form D/O acted with NAD+ as well as with O2 as electron acceptors, it exhibited greater affinity to NAD+ than to O2, and NAD+ abolished the oxidase activity of this form. Moreover, oxidase activity of form D/O was inhibited by NADH. These facts indicate that NAD+ and O2 compete for the same active site on the enzyme molecule.

| S-EPMC1161139 | biostudies-other

The presence of Xanthine Dehydrogenase is crucial for the maturation of the rat kidneys

Project description:We hypothesized that XDH plays a role in nephrogenesis. Here we used SSXdh+/+, SSXdh+/-, and SSXdh-/- rat kidneys from the postnatal development period to test this hypothesis. In wildtype but not homozygote rat kidneys, XDH expression was significantly increased from postnatal day (PND) 5 to 15, indicating its importance in the maturation of the kidney. Even though both genotypes had similar glomerular density at birth, it was significantly lower in SSXdh-/- rats by PND21. Transcriptomic profiling of the kidneys revealed that many genes related to kidney formation were dysregulated by the lack of Xdh expression in this period. Functional and pathway analyses of the transcriptome data strongly suggested that Xdh affected these genes via epidermal growth factor (EGF) signaling disruption. This study showed that Xdh is essential for the kidney maturation process.

2023-09-20 | GSE239437 | GEO

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