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At least 19 recordsLinked to original sources
PubMed1979

[Comparative study of chicken liver xanthine dehydrogenase and bovine liver xanthine oxidase. dehydrogenase activity of xanthine oxidase (author's transl)].

A method to purify bovine liver xanthine oxidase in described, with which samples of 256-fold specific activity with respect to the initial homogenate are obtained. Bovine liver xanthine oxidase and chicken liver xanthine dehydrogenase with oxygen as electron acceptor exhibit similar profile in pKM and log V versus pH plots. With NAD+ as electron acceptor a different profile in the pKM xanthine plot is obtained for chicken liver xanthine dehydrogenase. However three inflection points at the same pH values appear in all plots. Both enzymes are irreversibly inhibited by pCMB and reversibly by N-ethylmaleimide and by iodoacetamide, with competitive and uncompetitive type inhibitions respectively. These results suggest that NAD+ alters the enzymatic action since its binding to the enzyme antecedes the binding of xanthine to the xanthine oxidase molecule, without undergoing itself any modification. 0.15 M DDT of DTE treatment of bovine liver xanthine oxidase gives to the enzyme a permanent activity with NAD+ without modifying its activity with oxygen. The enzyme thus treated produces parallel straight lines in Lineweaver-Burk plots.

Animals↗
PubMed1978

Bovine milk xanthine oxidase: purification by ultrafiltration and conventional methods which omit addition of proteases: some criteria for homogeneity of native xanthine oxidase.

Methodological difficulties have been encountered when proteases were omitted from the conventional isolation of bovine milk xanthine oxidase (xanthine:oxygen oxidoreductase, EC 1.2.3.2). The use of these conventional methods has been studied and modified to reduce the problems encountered. Some of the difficulties may be due to the presence of high concentrations of caseins, which exhibit a wide range of charges and sizes, thereby making separations based on charge and size more complicated. In addition, non-covalent interactions may occur between the caseins and xanthine oxidase leading to the formation of casein-xanthine oxidase micellar aggregates. The difficulties encountered in this conventional isolation have been circumvented by purifying the enzyme directly from milk fat globule membranes that first have been washed free of most casein and other milk proteins. The xanthine oxidase is isolated by ultrafiltration through an Amicon XM-100A membrane at 5 degrees C in 0.25 M sucrose/5 mM sodium salicylate. The largest molecular size of globular proteins which can penetrate this ultrafiltration membrane has been previously estimated to be around 100 000 daltons. Xanthine oxidase thus appears to be smaller than 100 000 daltons in its native state. The size observed for active xanthine oxidase previously isolated by other methods has been around 275 000--300 000 daltons. Xanthine oxidase isolated by ultrafiltration appears similar to xanthine oxidase from conventional isolation methods according to empirical criteria of homogeneity based on size and also on the absorbances at 280 and 450 nm. Criteria based on charge were found to be less reliable.

Ammonium Sulfate↗
PubMed1976

Oxidation of N-methyl substituted hypoxanthines, xanthines, purine-6,8-diones and the corresponding 6-thioxo derivatives by bovine milk xanthine oxidase.

1. The oxidation of six series of purines (hypoxanthines, xanthines, purine-6,8-diones and the corresponding 6-thioxo derivatives) by a highly purified bovine milk xanthine oxidase (EC 1.2.3.2) has been studied, using a variety of N-methyl derivatives. 2. N-Methyl substituents can either enhance or reduce enzymic rates. Enhancement is ascribed to blockade of groups which mediate unfavorable modes of binding of substrate to enzyme. Introduction of N-methyl groups can also inhibit enzymic oxidation, either by occluding essential binding groups or by preventing spontaneous or enzyme-induced tautomerisation processes, which create suitable binding sites in the substrates. 3. In all purines which are rapidly attacked by xanthine oxidase, proper attachment to the active center is mediated by the groupings (3) NH, (9) N or (3) N, (9) NH. 4. Reduced rates usually express lowered substrate affinity, which finds its expression in weak competitive inhibition of xanthine oxidation.

Animals↗
PubMed1978

Magnetic coupling of the molybdenum and iron-sulphur centres in xanthine oxidase and xanthine dehydrogenases.

Magnetic interaction between molybdenum and one of the iron-sulphur centres in milk xanthine oxidase [Lowe, Lynden-Bell & Bray (1972) Biochem. J. 130, 239-249] was studied further, with particular reference to the newly discovered Mo(V) e.p.r.(electron-paramagnetic-resonance) signal, Resting II [Lowe, Barber, Pawlik & Bray (1976) Biochem. J. 155, 81-85]. E.p.r. measurements at 35GHz near to 4.2K showed that the interaction has the same sign at all molybdenum orientations and is ferromagnetic. The predicted splitting of the e.p.r. signal from the reduced iron-sulphur centre, Fe/S I, was observed, Providing positive identification of this as the other interacting species. Chemical modification of the molybdenum environment in xanthine oxidase can change the size of the interaction severalfold, but interaction always remains approximately isotropic. The interaction in turkey liver xanthine dehydrogenase is indistinguishable from that in the oxidase. However, a bacterial xanthine dehydrogenase with different iron-sulphur centres shows rather larger interaction. Guanidinium chloride disturbs the iron-sulphur centres of the oxidase, and when this occurs there is a parallel and relatively small change in the interaction. Removal of flavin from the molecule, or raising the pH to 12.0, changes the interaction slightly without affecting the chromophores themselves. It is concluded that the Fe/S I centre and the Mo are at least 1.0nm and probably nearer 2.5nm apart, and that the conformation of the protein between them is relatively stable up to pH 12.

Animals↗
PubMed1978

Electron-paramagnetic-resonance spectroscopy of complexes of xanthine oxidase with xanthine and uric acid.

Molybdenum(V) e.p.r. signals from reduced functional milk xanthine oxidase molecules (the Rapid signals), obtained in the presence of purine substrates and products, were further investigated [cf. Bray & Vänngård, (1969) Biochem. J. 114, 725-734; Pick & Bray (1969) Biochem. J. 114, 735-742]. Xanthine forms two complexes with the enzyme that are believed to correspond to different orientations of the substrate molecule in the active site. Only one complex appears to undergo the catalytic reaction. Non-productive complexes, analogous to theone with xanthine, are not formed by 1-methylxanthine or purine. Uric acid forms more than one e.p.r.-detectable complex, one of which is analogous to the non-productive xanthine complex. The computer program used for handing the e.p.r. data is described briefly.

Animals↗
PubMed1975

Nutritional control of xanthine dehydrogenase. II. Effects on xanthine dehydrogenase and aldehyde oxidase of culturing wild-type and mutant Drosophila on different levels of molybdenum.

Two new mutants, deficient in aldehyde oxidase and xanthine dehydrogenase, have been isolated from a wild-type stock of Drosophila melanogaster and have been provisionally termed lxd-c and lxd-d, respectively, as both mutants appear to be allelic with lxd (low xanthine dehydrogenase). An analysis has been made of the effects of dietary molybdenum on lxd, lxd-c, lxd-d, lao (low aldehyde oxidase), mal (maroon-like eye color), and pac (Pacific) wild-type flies. On the lower dietary levels of 10(-3) M and 10(-2) M molybdenum, increases in specific activity of both enzymes were observed only in lxd. Furthermore, two- to three-fold increases in specific activity of both enzymes occurred in all strains, except mal, when cultured on 5 x 10(-2) M molybdenum. The lxd and lxd-c strains failed to survive on this high concentration of the ion. Similar concentrations of molybdenum had no effect in vitro. An extra electrophoretic band of xanthine dehydrogenase was observed on polyacrylamide gel from extracts of wild-type flies cultured on certain levels of molybdenum, but its appearance was not always correlated with the increases in specific activity.

Aldehyde Oxidoreductases↗
PubMed1978

pH-jump studies at subzero temperatures on an intermediate in the reaction of xanthine oxidase with xanthine.

Xanthine oxidase is stable and active in aqueous dimethyl sulphoxide solutions of up to at least 57% (w/w). Simple techniques are described for mixing the enzyme in this solvent at--82 degrees C, with its substrate, xanthine. When working at high pH values under such conditions, no reaction occurred, as judged by the absence of e.p.r. signals. On warming to--60 degrees C, for 10 min, however, the Very Rapid molybdenum(V) e.p.r. signal was obtained. This signal did not change on decreasing the pH, while maintaining the sample in liquid nitrate reductase, caused its molybdenum(V) e.p.r. signal to change from the high-pH to the low-pH form. These findings are not compatible with the conclusions of Edmondson, Ballou, Van Heuvelen, Palmer & Massey [J. Biol. Chem. (1973) 248, 6135-6144], that the Very Rapid signal is in prototropic equilibrium with the Rapid signal, and should be important in understanding the mechanism of action of the enzyme. They emphasize the unique nature of the intermediate represented by the Very Rapid e.p.r. signal. The possible value of the pK for loss of an exchangeable proton from the Rapid signal is discussed.

Chemical Phenomena↗
PubMed1977

Distribution of xanthine oxidase and xanthine dehydrogenase specificity types among bacteria.

A diverse collection of xanthine-metabolizing bacteria was examined for xanthine-, 1-methylxanthine-, and 3-methylxanthine-oxidizing activity. Both particulate and soluble fractions of extracts from aerobically grown gram-negative bacteria exhibited oxidation of all three substrates; however, when facultative gram-negative bacteria were grown anaerobically, low particulate and 3-methylxanthine activities were detected. Gram-positive and obligately anaerobic bacteria showed no particulate activity or 3-methylxanthine oxidation. Substrate specificity studies indicate two types of enzyme distributed among the bacteria along taxonomic lines, although other features indicate diversity of the enzyme within these two major groups. The soluble and particulate enzymes from Pseudomonas putida and the enzyme from Arthrobacter S-2 were examined as type examples with a series of purine and analogues differing in the number and position of oxygen groups. Each preparation was active with a variety of compounds, but the compounds and position attacked by each enzyme was different, both from the other enzymes examined and from previously investigated enzymes. The soluble enzyme from Pseudomonas was inhibited in a competitive manner by uric acid, whereas the Arthrobacter enzyme was not. This was correlated with the ability of Pseudomonas, but not Arthrobacter, to incorporate radioactivity from [2-14C]uric acid into cellular material.

Actinomycetales↗
PubMed1979

On the biochemical mechanism of action of 1-propyl-3-methyl-7-(5-hydroxy-hexyl)-xanthine (HWA 153), a new bronchospasmolytically active methyl xanthine derivative.

1-Propyl-3-methyl-7-(5-hydroxy-hexyl)-xanthine (HWA 153) is a new bronchospasmolytic agent with a significant influence on the cAMP system of lungs and bronchi. In in vitro experiments HWA 153 inhibits cAMP phosphodiesterase (PDE) isolated from bovine bronchi more than does theophylline. HWA 153 is (in conc. 5 x 10(-4) mol/l) 1.8 and 4.3 times more active as a PDE inhibitor of guinea pig lungs and bronchi, respectively, than theophylline-ethylenediamine. HWA 153 also stabilizes rat erythrocyte membrane against hypoosmotic shock. In isolated guinea pig bronchi HWA 153 (in conc. 5 x 10(-4) mol/l) decreases by 77% bronchial spasm induced by the addition of histamine (5 x 10(-5) mol/l). A significant increase in cAMP level of bronchi was simultaneously observed. In in vivo experiments HWA 153 (25 mg/kg p.o.) inhibits PDE of lungs and bronchi of guinea pigs. Simultaneously, a significant increase in cAMP level in these organs was observed. In in vivo experiments with hypoxic rats, HWA 153 (25 mg/kg p.o.) increases ATP, ATP/ADP ratio and adenylate energy charge (AEC) in hypoxic rats, 1 h after administration. This indicates a positive influence of HWA 153 on the energy metabolism of red blood cells.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1979

Kinetics and stability of immobilized chicken liver xanthine dehydrogenase.

Xanthine dehydrogenase (EC 1.2.1.37) was isolated from chicken livers and immobilized by adsorption to a Sepharose derivative, prepared by reaction of n-octylamine with CNBr-activated Sepharose 4B. Using a crude preparation of enzyme for immobilization it was observed that relatively more activity was adsorbed than protein, but the yield of immobilized activity increased as a purer enzyme preparation was used. As more activity and protein were bound, relatively less immobilized activity was recovered. This effect was probably due to blocking of active xanthine dehydrogenase by protein impurities. The kinetics of free and immobilized xanthine dehydrogenase were studied in the pH range 7.5-9.1. The Km and V values estimated for free xanthine dehydrogenase increase as the pH increase; the K'm and V values for the immobilized enzyme go through a minimum at pH 8.1. By varying the amount of enzyme activity bound per unit volume of gel, it was shown that K'm is larger than Km are result of substrate diffusion limitation in the pores of the support material. Both free and immobilized xanthine dehydrogenase showed substrate activation at low concentrations (up to 2 microM xanthine). Immobilized xanthine dehydrogenase was more stable than the free enzyme during storage in the temperature range of 4-50 degrees C. The operational stability of immobilized xanthine dehydrogenase at 30 degrees C was two orders of magnitude smaller than the storage stability, t 1/2 was 9 and 800 hr, respectively. The operational stability was, however, better than than of immobilized milk xanthine oxidase (t 1/2 = 1 hr). In addition, the amount of product formed per unit initial activity in one half-life, was higher for immobilized xanthine dehydrogenase than for immobilized xanthine oxidase. Unless immobilized milk xanthine oxidase can be considerable stabilized, immobilized chicken liver xanthine dehydrogenase is more promising for application in organic synthesis.

Animals↗
PubMed1976

Oxidation of selected pteridine derivatives by mamalian liver xanthine oxidase and aldehyde oxidase.

Considerable information is available concerning the oxidation of pteridine derivatives by bovine milk xanthine oxidase, but few investigations have been carried out on the oxidation of such compounds by mammalian liver xanthine oxidase and the related aldehyde oxidase. Xanthine oxidase, obtained from rat liver, oxidizes a variety of substituted amino- and hydroxypteridines in a manner identical to that previously observed for milk xanthine oxidase. For example, 2-aminopteridine and its 4- and 7-hydroxy derivatives were oxidized efficiently to 2-amino-4,7-dihydroxypteridine (isoxanthopterin) by the rat liver enzyme, and 4-aminopteridine and its 2- and 7-hydroxy derivatives were oxidized to 4-amino-2,7-dihydroxypteridine.4-Hydroxypteridine and the isomeric 2- and 7-hydroxypteridines were oxidized by rat liver xanthine oxidase to 2,4,7-trihydroxypteridine. Rabbit liver aldehyde oxidase, but not rat liver xanthine oxidase, was able to catalyze the oxidation in position 7 of 2,4-diaminopteridine and its 6-methyl and 6-hydroxymethyl derivatives. 2-Aminopteridine and 4-aminopteridine were both oxidized to the corresponding 7-hydroxy derivatives in the aldehyde oxidase system; 2-amino-4-hydroxypteridine appeared to be a minor product in the oxidation of 2-aminopteridine by rabbit liver aldehyde oxidase. Both aldehyde oxidase and xanthine oxidase were able to catalyze the oxidation of 2-amino-6,7-disubstituted pteridines to the corresponding 4-hydroxy derivatives; 4-hydroxy-6,7-disubstituted pteridines were oxidized in position 2 by both enzymes. 4-Amino-6,7-disubstituted pteridines were not oxidized by either enzyme. 2-Amino-4-methylpteridine was oxidized in position 7 by aldehyde oxidase but was not an effective substrate for xanthine oxidase; 2-hydroxypteridine and 7-hydroxypteridine were not oxidized to a detectably extent by aldehyde oxidase. All oxidations mediated by xanthine oxidase were strongly inhibited by allopurinol (4-hydroxypyrazolo[3,4-d]pyrimidine), and all oxidations mediated by aldehyde oxidase were inhibited by menadione (2-methyl-1,4-naphthoquinone). Rat liver xanthine oxidase and, to a lesser extent, rabbit liver aldehyde oxidase were inhibited by 4-chloro-6,7-dimethylpteridine; 2-amino-3-pyrazinecarboxylic acid inhibited xanthine oxidase but not aldehyde oxidase. The oxidations of 2- and 4-aminopteridines by aldehyde oxidase resulted in concomitant reduction of cytochrome c.

Aldehyde Oxidoreductases↗
PubMed1978

Regulation, purification, and properties of xanthine dehydrogenase in Neurospora crassa.

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.

Enzyme Induction↗
PubMed1977

Bovine milk xanthine oxidase, blood lipids and coronary plaques in rabbits.

The effects of prolonged intravenous administration of bovine milk xanthine oxidase (EC 1.2.3.2.) on blood lipids and arterial integrity were measured to determine if the administration of this enzyme produces metabolic changes conducive to plaque formation. New Zealand White rabbits were injected intravenously with bovine milk xanthine oxidase at 4-day intervals during a 13-week test period. At the end of the test period, the rabbits were killed and blood, heart, aorta, liver, and kidneys were collected and evaluated. Rabbits injected with phosphate buffer or acid-denatured xanthine oxidase for the same length of time served as negative controls. Additional rabbits fed a diet containing 3% added cholesterol for the same time period served as positive controls. The administration of xanthine oxidase in large amounts over a prolonged period did not alter serum cholesterol or triglyceride levels and did not reduce plasmalogen levels in the aorta or heart. Xanthine oxidase administration did not induce arterial plaque formation. Cholesterol feeding over the same time period increased serum cholesterol levels, reduced liver xanthine oxidase activity levels and resulted in a marked development of arterial plaques. Althouth xanthine oxidase activity was found in liver from all rabbits, enzyme activity was not detectable in aorta, heart or kidneys from any rabbit. Free or complexed bovine milk xanthine oxidase could not be demonstrated in heart, aorta, liver or kidneys from any of the rabbits with immunodiffusion or with immunofluorescent techniques. The study showed that when large intravenous doses of bovine milk xanthine oxidase were given to rabbits, the enzyme was not deposited in heart, aorta, liver or kidneys. The study also showed that large intravenous doses of xanthine oxidase over prolonged periods did not deplete arterial or coronary tissue plasmalogens, and did not induce arterial plaque formation.

Animals↗
PubMed1978

Superoxide-independent platelet response to xanthine oxidase.

Xanthine oxidase (1--5 microgram/ml) from cow's milk induces shape change, aggregation, and the release reaction of human washed platelets. Xanthine oxidase plus xanthine produce superoxide radicals, which reduce nitro blue tetrazolium. Superoxide dismutase, allopurinol, or ommission of xanthine inhibits the reduction of nitro blue tetrazolium but has no influence on the platelet response to xanthine oxidase. In contrast, small amounts of plasma or apyrase from potatoes abolish the effect on platelets, but not the enzyme activity of xanthine oxidase. Comparison of two xanthine oxidase preparations shows that higher specific enzyme activity corresponds to a lesser effect on platelets. The results suggest that platelet and enzyme activities reside in different components of xanthine oxidase preparations.

Apyrase↗