Chicken Liver Xanthine Dehydrogenase Research Articles

Chicken liver xanthine dehydrogenase kinetic behaviour with oxygen or NAD + as electron acceptors

The behaviour of chicken liver xanthine dehydrogenase (xanthine: NAD +—oxidoreductase (E.C. 1.2.1.37)) with oxygen or with NAD + as electron acceptors is compared. With oxygen, the double reciprocal plot of v versus [xanthine] 0.5 is linear, whereas for NAD + the 1/ v versus 1/[xanthine] plots are linear. With NAD + as substrate (xanthine:NAD +—oxidoreductase activity or with NADH (diaphorasic activity)), the double reciprocal plots of v versus [substrate] are linear. The pattern of uric acid inhibition of the xanthine dehydrogenase against xanthine varies with the electron acceptor considered. The kinetic constants characterising the systems have been determined.

Electron paramagnetic resonance properties and oxidation-reduction potentials of the molybdenum, flavin, and iron-sulfur centers of chicken liver xanthine dehydrogenase

The oxidation-reduction potentials of the various prosthetic groups in the native and desulfo forms of chicken liver xanthine dehydrogenase, determined by potentiometric titration in 0.05 m potassium phosphate buffer, pH 7.8, are: Mo(VI)/Mo(V) (native), −357 mV; Mo(VI)/Mo(V) (desulfo), −397 mV; Mo(V)/Mo(IV) (native), −337 mV; Mo(V)/Mo(IV) (desulfo), −433 mV; FAD/FADH · −345 mV; FADH · FADH 2, − 377 mV; (Fe/S)I ox/(Fe/S)I red, −280 mV; (Fe/S)II ox/(Fe/S)II red, − 275 mV. Titration at pH 6.8 revealed that the Mo and FAD centers but not the Fe/S centers are in prototropic equilibrium. Spectroscopic studies on the native and deflavinated enzymes show that environment of the flavin in xanthine dehydrogenase differs from that in bovine milk xanthine oxidase.

Isolation of the domain containing the molybdenum, iron-sulfur I, and iron-sulfur II centers of chicken liver xanthine dehydrogenase.

Chicken liver xanthine dehydrogenase, like other xanthine-oxidizing enzymes, is a dimer of Mr = 150,000 subunits. Each subunit contains one molybdenum, one FAD, and two distinct Fe2S2 centers. Treatment with a number of proteases shows that the native enzyme subunit is cleaved at three distinct sites. However, the cleavage products can be separated only under denaturing conditions. Prolonged treatment with subtilisin at pH 10.1 has permitted the isolation of an Mr = 65,000 catalytically active fragment that is devoid of FAD but which contains the molybdenum and both types of iron-sulfur center. A model of the domain structure of the native enzyme is proposed.

Syncatalytic modification of chicken liver xanthine dehydrogenase by hydrogen peroxide. The nature of the reaction.

Xanthine dehydrogenase engaged in catalyzing the oxidation of substrate by oxygen is repidly inactivated by the hydrogen peroxide generated during the reaction. Experimental evidence shows that peroxide reacts more readily with the reduced than with the oxidized form of the enzyme. Inactivation results from modification of the cyanolysable sulfur present at the molybdenum center.

Uric acid as electron acceptor of liver xanthine dehydrogenase

Abstract The stoichiometry of the hypoxanthine + uric acid → 2 xanthine reaction, catalysed by chicken liver xanthine dehydrogenase (xanthine: NAD+-oxidoreductase (E.C. 1.2.1.37)), has been determined by u.v. spectrophotometry. The system is adapted to a ping-pong type mechanism; in the proposed reaction sequence, the hypoxanthine is oxidised to xanthine and the reduced form of the enzyme is oxidised by the uric acid, whereby a second xanthine molecule is formed. The reaction product, xanthine, is a non-competitive inhibitor of the enzyme relative to both substrates. Hypoxanthine and excess uric acid are competitive, linear inhibitors of the system.

An immunochemical study of the changes in chicken liver xanthine dehydrogenase activity during dietary adaptation.

A specific rabbit antiserum was prepared against chicken liver xanthine dehydrogenase [EC 1.2.1.37] and used to investigate changes in the amount of the enzyme protein in the liver during dietary adaptation. When the enzyme activity was decreased by the administration of a low protein diet, quantitative immunoprecipitation and Laurell electroimmunoassay showed not only a decrease in the amount of the enzyme antigen but also a reduction of the apparent molecular activity of the enzyme. This was confirmed by the finding that the activity-flavin ratio and the activity-molybdenum ratio of the enzyme were lowered in chickens adapted to the low protein diet. The relative rate of synthesis of xanthine dehydrogenase, expressed as radioactivity incorporated into the enzyme protein divided by radioactivity incorporated into total cell homog-enate protein, was measured by pulse-labeling the liver protein with L-[4,5−33H]leucine. Xanthine dehydrogenase was isolated by quantitative immunoprecipitation followed by electrophoresis of the dissolved immunoprecipitate on sodium dodecyl sulfate-polyacrylamide gel. The relative rate of synthesis increased 6– to 9–fold, while the enzyme activity increased 10– to 30–fold in vivo following the administration of a high protein diet. The rate of degradation of xanthine dehydrogenase was higher in chickens adapted to the low protein diet than in those adapted to the high protein diet, the apparent half lives being 1 day and 3 days, respectively. Although some limitations and assumptions were present in the techniques used in these experiments, these results suggest that the increase in the hepatic xanthine dehydrogenase content in the chickens adapted to the high protein diet was mainly due to a rise in the rate of enzyme synthesis and, to a minor degree, to a decrease in the rate of enzyme degradation.

The Interaction of Arsenite with the Molybdenum Center of Chicken Liver Xanthine Dehydrogenase

Inactivation of chicken liver xanthine dehydrogenase by arsenite is reflected in the molybdenum electron paramagnetic resonance signal at g = 1.97. The arsenite spectrum shows additional splittings and considerable broadening yet remains comparable to the native in total intensity. Further subtle alterations of the molybdenum signal of arsenite-treated enzyme are seen in the presence of purine-type substrates or inhibitors.

Purification and properties of the NAD +-dependent (type D) and O 2-dependent (type O) forms of rat liver xanthine dehydrogenase

The xanthine-oxidizing enzyme of rat liver has been purified as an NAD +-dependent dehydrogenase (type D) and as the O 2-dependent oxidase (type O). The purified D and O variants are nearly homogenous as judged by polyacrylamide discontinuous gel electrophoresis and are indistinguishable on sodium dodecyl sulfate-urea gels. The absorption spectrum of the type D enzyme is indistinguishable from that of the type O enzyme and closely resembles the spectra of xanthine-oxidizing enzymes from other sources. The types D and O enzymes have essentially the same cofactor composition. Oxidation of xanthine by type D is stimulated by NAD + with concomitant NADH formation. Type D is able to utilize NADH as well as xanthine as electron donor to various acceptors, in contrast to type O that is unable to oxidize NADH. Arsenite, cyanide and methanol completely abolish xanthine oxidation by the type D enzyme while affecting the activities with NADH to varying extents. In these respects rat liver xanthine dehydrogenase closely resembles chicken liver xanthine dehydrogenase. However, in contrast to the avian enzyme, the purified rat liver enzyme is unstable as a dehydrogenase and is gradually converted to an oxidase. This conversion is accompanied by an increase in the aerobic xanthine → cytochrome c activity. The native type D enzyme in rat liver extracts is precipitable with antibody prepared against purified type O. The K m for xanthine is not significantly different for the two forms.

Studies on chicken liver xanthine dehydrogenase with reference to the problem of non-equivalence of FAD moieties

1. 1. Reduction of chicken liver xanthine dehydrogenase (xanthine : NAD + oxidoreductase, EC 1.2.1.37) by xanthine under anaerobic condition proceeded in two phases. This biphasicity may be due to functional and non-functional enzymes in the enzyme preparation. 2. 2. Cyanolysis of a persulfide group of chicken liver enzyme resulted in an inactivation of the enzyme. The non-functional enzyme in the standard enzyme preparation was found to lack persulfide groups at the active sites. 3. 3. The remaining NADH-Methylene Blue oxidoreductase activity, after KI treatment of the xanthine-reduced enzyme of a high flavin activity ratio, is not at the level of 50% of the initial activity, differing from the report suggesting non-equivalence of FAD chromophores. 4. 4. The findings in the present report indicate that FAD chromophores of chicken liver enzyme are essentially equivalent.

Involvement of Molybdenum and Iron in the in Vitro Assembly of Assimilatory Nitrate Reductase Utilizing Neurospora Mutant nit-1

Abstract Addition of high levels of sodium molybdate (10-2 m, final concentration) to acid-treated purified bovine milk xanthine oxidase prior to incubation with an extract of the nitrate-induced nitrate reductase Neurospora crassa mutant nit-1 resulted in a significant enhancement of the subsequent in vitro assembly of wild type N. crassa NADPH-nitrate reductase (EC 1.6.6.2). Added molybdate had no effect on the wild type enzyme itself. The enhancement is highly specific for molybdenum, is independent of added phosphate, and also occurs with cell-free preparations of uninduced wild type and with other known acid-treated molybdenum enzymes including chicken liver xanthine dehydrogenase, rabbit liver aldehyde oxidase, bovine sulfite oxidase, and the nitrogenases of Azotobacter and Clostridium. Preincubation at 23° of acid-treated xanthine oxidase preparations resulted in a considerable loss of ability to form nitrate reductase and in a more striking enhancement by subsequently added molybdate. Prior addition of molybdate during the above 23° preincubation prevented in part the loss of the ability of the enzyme to form nitrate reductase, suggesting that the enhancement by molybdate is a restoration effect rather than a stimulatory one. Inclusion of molybdate in the buffer used for homogenization of the uninduced wild type also gave a significant increase in the subsequent in vitro assembly of the enzyme. Various forms and derivatives of molybdenum enhanced to varying degrees the ability of acid-treated xanthine oxidase to form nitrate reductase. Aqueous solutions of several complexes of Mo(V) with cysteine or glutathione under anaerobic conditions failed to substitute for the presumed molybdenum cofactor in the in vitro assembly of nitrate reductase. Sucrose density gradient profiles of NADPH-nitrate reductase formed in vitro by incubation of induced nit-1 extract with a 99Mo-labeled fraction of uninduced wild type (grown on 99MoO4=-containing medium) coincided with that of radioactivity. The addition of 99MoO4= to a cell-free preparation of unlabeled uninduced wild type or to acid-treated xanthine oxidase prior to its incubation with an extract of induced nit-1 gave similar results. Reciprocal experiments using an unlabeled fraction of uninduced wild type and an extract of nit-1 grown and induced in the presence of 99MoO4= showed no incorporation of radioactivity in the in vitro assembled enzyme. Nor was there in vitro exchange or incorporation of 99Mo into the already formed wild type enzyme. Comparable experiments with 55Fe showed that nit-1 and not uninduced wild type or xanthine oxidase is the source of iron in the in vitro assembly of Neurospora NADPH-nitrate reductase.

Synthesis and degradation of chicken liver xanthine dehydrogenase during development.

Abstract The rate of synthesis and degradation of xanthine dehydrogenase in 14-day-old chick embryos, 1-day-old and 8-day-old chicks was determined. When [ 14 C]arginine is used the rate constant for synthesis is low in the embryo, increases about 4-fold immediately upon hatching, and reaches a maximum by the eighth day after hatching. The rate constant when [ 14 C]valine is the substrate is 2.5 times greater in the 1-day-old chick than the value obtained with arginine. Degradation of xanthine dehydrogenase is low in the embryo, or the 1-day-old chick, but increases in the 8-day-old chick.

Mechanism of action of a respiratory inhibitor from the gill tissue of the sporulating common mushroom, Agaricus bisporus

γ- l-glutaminyl 3,4-benzoquinone, isolated from the gill tissue of speculating common meadow mushroom Agaricus bisporus has been found to inhibit the dehydrogenases of the tricarboxylic acid cycle in rat liver mitochondria. Inhibition of succinic-cytochrome c reductase activity is competitively retarded by succinate and malonate. Studies on the inhibition of chicken liver xanthine dehydrogenase indicated that the quinone is a sulfhydryl blocking reagent. Several other “sulfhydryl” enzymes are also inhibited by the quinone. Formation of a 1:1 adduct with cysteine was detected spectrophotometrically. Similar adducts are formed on treatment of “sulfhydryl” enzymes with the quinone.

Nonequivalence of the Flavin Adenine Dinucleotide Moieties of Chicken Liver Xanthine Dehydrogenase

Abstract Prior reduction of chicken liver xanthine dehydrogenase with xanthine or NADH leads to accelerated loss of enzymic FAD on exposure to 3 m KI. Reduction with NADH leads to the dissociation of all of the FAD, whereas reduction with xanthine results in the rapid dissociation of only 50% of the FAD. In both cases the xanthine → NAD activity is completely abolished. Oxidation of xanthine in the presence of artificial electron acceptors is effected at comparable rates by the two apoproteins. Oxidation of NADH by the partially deflavinated enzyme is generally commensurate with its flavin content, whereas the fully deflavinated enzyme is incapable of oxidizing NADH. Spectrophotometric and electron paramagnetic resonance studies on the apoproteins have shown that the partially deflavinated enzyme is reducible by both xanthine and NADH, whereas the fully deflavinated enzyme is reducible only by xanthine. The results are discussed in the context of the arrangement of the electron carrier components in the active center of the enzyme.

Studies on the Dissociation of Flavin Adenine Dinucleotide from Metalloflavoproteins

Abstract Treatment of milk xanthine oxidase and chicken liver xanthine dehydrogenase with high concentrations of salts for several hours resulted in the loss of reactivity toward O2 and NAD, respectively. Oxidation of xanthine by either enzyme in the presence of electron acceptors such as dichlorophenol indophenol was unimpaired under these conditions, in the presence of 3 m KI. Prolonged treatment of xanthine oxidase and xanthine dehydrogenase with 3 m KI followed by extensive dialysis in 0.5 m KCl yielded flavin-free metalloproteins which had the absorption characteristics of their iron-sulfur chromophore. Both flavin-free enzymes showed markedly enhanced rates of electron transfer to cytochrome c and nitroblue tetrazolium compared to the native enzymes. The cytochrome c activity of flavin-free xanthine oxidase, in contrast to the same activity of native xanthine oxidase, was completely insensitive to superoxide dismutase. Prior reduction of the enzymes with their electron donor substrates resulted in a dramatic increase in the lability of FAD in the presence of 3 m KI. Thus, in the case of xanthine dehydrogenase reduced with NADH, the FAD was nearly completely dissociated in less than 5 min compared to the several hours required with the native enzyme. The flavin-free enzymes prepared by the modified procedure were much more stable, and showed superior ability to be reconstituted with FAD.

Sythesis and degradation of chicken liver xanthine dehydrogenase during development

The rate of synthesis and degradation of xanthine dehydrogenase in 14-day-old chick embryos, 1-day-old and 8-day-old chicks was determined. When [ 14C]arginine is used the rate constant for synthesis is low in the embryo, increases about 4-fold immediately upon hatching, and reaches a maximum by the eighth day after hatching. The rate constant when [ 14C]valine is the substrate is 2.5 times greater in the 1-day-old chick than the value obtained with arginine. Degradation of xanthine dehydrogenase is low in the embryo, or the 1-day-old chick, but increases in the 8-day-old chick.

Nonlinear kinetics of the xanthine oxidase reaction catalyzed by chicken liver xanthine dehydrogenase

The xanthine oxidase reaction catalyzed by chicken liver xanthine dehydrogenase has been shown to give nonlinear kinetics of the type which has been identified as substrate activation. When a very wide range of substrate (pteridine) concentrations were studied, it was found that a downward deflection in reciprocal plots (substrate activation) occurs in the high region and an upward deflection in the very low region. When product (isoxanthopterin) was included in reaction mixtures, the upward deflection was enhanced and shifted to higher substrate concentration ranges. In addition, reciprocal plots with a second substrate (oxygen) and a product (isoxanthopterin) were nonlinear. On the basis of these results an empirical rate equation was deduced from a series of slope and intercept plots. Kinetic constants were evaluated by the method of weighted least squares using an IBM 360 digital computer. The standard error of the calculated reciprocal velocity was only 4.7% of the weighted average of the observed reciprocal velocity. No term in this empirical equation could be omitted or rearranged and attain the same degree of agreement. This nonlinear rate equation can be interpreted using random substrate addition and product-release kinetic models with independent catalytic sites and without postulating second sites.

In vitro assembly of Neurospora assimilatory nitrate reductase from protein subunits of a Neurospora mutant and the xanthine oxidizing or aldehyde oxidase systems of higher animals.

In vitro assembly or complementation of a hybrid assimilatory nitrate reductase was attained by mixing a preparation of nitrate-induced N. crassa mutant nit-1 specifically with acid-treated (pH 2.5) bovine milk or intestinal xanthine oxidase, rabbit liver aldehyde oxidase, or chicken liver xanthine dehydrogenase. The complementation reaction specifically required induced nit-1, the only nitrate reductase mutant of Neurospora that lacked xanthine dehydrogenase and was unable to use hypoxathine or nitrate as a sole nitrogen source. The complementing activities of the above acid-treated enzymes correspond to their xanthine or aldehyde oxidizing activity profiles on sucrose density gradients. The resulting soluble, reduced nicotinamide adenine dinucleotide phosphate (NADPH)-nitrate reductases are the same as the Neurospora wild type enzyme in sucrose density gradient profile, molecular weight, substrate affinities, and sensitivity to inhibitors and temperature. By analogy to a similar in vitro complementation of nitrate reductase in mixtures of induced nit-1 and individual nonalleic Neurospora mutants, or uninduced wild type, the complemented nitrate apparently consists of an inducible protein subunit (possessing inducible NADPH-cytochrome c reductase) furnished by nit-1 and a subunit from the acid-treated xanthine or aldehyde oxidizing system which can substitute for the constitutive component furnished by the other mutants or uninduced wild type. The data suggest that Neurospora nitrate reductase and the xanthine oxidizing system and aldehyde oxidase of animals, all of which are molybdenum-containing enzymes catalyzing the reduction of nitrate to nitrite, share a highly similar protein subunit.

The Utility of Superoxide Dismutase in Studying Free Radical Reactions: II. THE MECHANISM OF THE MEDIATION OF CYTOCHROME c REDUCTION BY A VARIETY OF ELECTRON CARRIERS

Abstract The rate of reduction of cytochrome c by the ferredoxin-TPN reductase of spinach or by chicken liver xanthine dehydrogenase is markedly augmented by a variety of electron carriers. Menadione, flavin mononucleotide, flavin adenine dinucleotide, N-methylphenazinium methosulfate, methylene blue, and 2,6-dichlorobenzenone indophenol were all effective. When cytochrome c reduction was mediated aerobically by menadione, flavin mononucleotide, or flavin adenine dinucleotide, superoxide dismutase was an inhibitor. When cytochrome c reduction was mediated by these same electron carriers in the absence of molecular oxygen, superoxide dismutase was without effect. When cytochrome c reduction was mediated by N-methylphenazinium methosulfate, methylene blue, or 2,6-dichlorobenzenone indophenol, superoxide dismutase was without effect whether or not oxygen was present. These results indicate that of these electron carriers only the reduced forms of menadione and of the flavins preferentially reduce O2 to the superoxide radical in the presence of cytochrome c. In the absence of cytochrome c, it was shown that reduced methylene blue is likewise capable of the univalent reduction of oxygen. In this case O-2 was detected by its ability to oxidize epinephrine to adrenochrome.

Purification and Properties of Inosinic Acid Dehydrogenase from Escherichia coli

Abstract Inosinic acid dehydrogenase was prepared from a derepressed mutant of Escherichia coli. Although the reaction catalyzed formally resembles those catalyzed by chicken liver xanthine dehydrogenase and related enzymes, this enzyme is devoid of any flavin, iron complex, or molybdenum component, and the kinetics indicate necessity for concomitant binding of substrate and DPN. The enzyme is absolutely dependent on K+ or NH4+, which are competitive with the inhibitor Mg++. A series of purine nucleotides inhibits competitively with substrate with no evidence of cooperative interactions. The enzyme is quite unstable and can be separated into several distinct but enzymatically equivalent fractions by centrifugation or electrophoresis.

The Role of Molybdenum in Xanthine Oxidase and Related Enzymes: Reactivity with Cyanide, Arsenite, and Methanol

The binding of arsenite, cyanide, or methanol by chicken liver xanthine dehydrogenase, milk xanthine oxidase, and rabbit liver aldehyde oxidase results in characteristic alterations in the absorption spectra of these enzymes. The coincidence between the kinetics of inhibition of all three enzymes and the rate of development of characteristic difference spectra has been established. These difference spectra have afforded a useful tool for investigating the binding sites of these inhibitors. The results indicate that all three ligands bind to the molybdenum component at the active center of each enzyme. Electron paramagnetic resonance spectra of native and arsenite- and cyanide-treated xanthine dehydrogenase have provided more definitive evidence for the binding of these inhibitors to enzymic molybdenum.