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.     

In vitro effects of corticosteroids, DDT, and 4, 9-dichlorodibenzodioxin on rat liver xanthine oxidase activity. Interactions between xanthine oxidase and cytochrome P450 in rat liver in vivo

It was established that deoxycorticosterone, cortisol, dexamethasone, DDT, and 4,9-dichlorodibenzodioxin inhibit in vitro binding of xanthine to highly purified rat liver xanthine oxidase. They are suggested to be allosteric inhibitors. The corresponding inhibition and binding constants were estimated. Also established was that cortisol and DDT, in dose 20 mg per kg of weight, inhibit xanthine oxidase and increase microsomal cytochrome P450 level in rat liver. There is an inverse relationship between xanthine oxidase activity and cytochrome P450 level in rat liver.     

The different effects of the peroxisome proliferators clofibric acid and bis(2-ethylhexyl)phthalate on the activities of peroxisomal oxidases in rat liver

Treatment with peroxisome proliferators induces increased numbers and alterations in the shape of peroxisomes in liver. It ultimately leads to hepatocellular carcinomas induced by the persistent production of high amounts of H2O2 as a result of a dramatical increase in acyl-CoA oxidase activity. The effects of peroxisome proliferators on other peroxisomal oxidase activities are less well documented. In the present study, the distribution patterns of the activity of SdD-amino acid oxidase, SlD-alpha-hydroxy acid oxidase, polyamine oxidase, urate oxidase and catalase activities were investigated in unfixed cryostat sections of liver, kidney and duodenum of rats treated with either clofibrate or bis(2-ethylhexyl)phthalate. The activities of xanthine oxidoreductase, which produces urate, a potent anti-oxidant, and xanthine oxidase, which produces oxygen radicals, were studied as well. The liver was the only organ that was affected by treatment. The number of peroxisomes increased considerably. SdD-Amino acid oxidase and polyamine oxidase activities were completely abolished by the treatment, whereas SlD-alpha-hydroxy acid oxidase activity decreased and urate oxidase activity increased periportally and decreased pericentrally. Total catalase activity increased because of the larger numbers of peroxisomes, but it decreased per individual peroxisome. Xanthine oxidoreductase activity decreased, whereas the percentage of xanthine oxidase remained constant. We conclude that oxidases in rat liver are affected differentially, indicating that the expression of activity of each oxidase is regulated individually. © 1998 Chapman & Hall     

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.     

Developmental Changes in Rat Liver Xanthine Oxidase(s) and Its Intracellular Distribution

Developmental change and subcellular distribution of xanthine oxidase in the rat liver were examined.

The specific activity of the fetal liver xanthine oxidase increased sharply to the levels of the adult liver on the day of the birth. After birth, the activity dropped rapidly and on the 14th day after birth it was about 1/4 of adult level. Then the activity was regained and around 28th day after birth it was about the same as in adult level.

In the livers from 80 days old rats, about 60% of total xanthine oxidase activity was found in soluble fraction and the rest was distributed among particulate fractions including microsomal, lysosomal, mitochondrial and nuclear fractions.

In contrast to the adult livers 80% of total xanthine oxidase activity in fetal liver was found to be in particulate fractions.

From kinetic studies of xanthine oxidases in particulate and soluble fractions it was suggested that xanthine oxidase in soluble fraction and xanthine oxidase in particulate fraction might be different in their natures of protein molecule.     

Effect of delta sleep-inducing peptide on lipid peroxidation and xanthine oxidase activity in rat tissues during cold stress

I. p. administration of exogenous delta-sleep-inducing peptide (DSIP) decreased the amount of diene conjugates and Schiff bases in the liver and brain in rats. The xanthine oxidase activity, at that, did not change. Cold stress enhanced the xanthine oxidase activity well as the amount of diene conjugates and Schiff bases. Preliminary administration of the delta-sleep-inducing peptide to cold-exposed animals diminished the xanthine oxidase activity and lipid peroxidation in the liver and brain. Protective effects of the DSIP under stress is discussed.     

Absence of xanthine oxidase or xanthine dehydrogenase in the rabbit myocardium

We directly measured the activity of the enzymes xanthine oxidase and xanthine dehydrogenase in rabbit and rat hearts, using a sensitive radiochemical assay. Neither xanthine oxidase activity nor xanthine dehydrogenase activity was detected in the rabbit heart. In the rat heart, xanthine oxidase activity was 9.1 +/- 0.5 mIU per gram wet weight and xanthine dehydrogenase activity was 53.0 +/- 1.9 mIU per gram wet weight. These results argue against the involvement of the xanthine oxidase/xanthine dehydrogenase system as a mechanism of tissue injury in the rabbit heart, and suggest that the ability of allopurinol to protect the rabbit heart against hypoxic or ischemic damage must be due to a mechanism other than inhibition of these enzymes.     

Diamine oxidase activity induction in regenerating rat liver

The synthesis and turnover of diamine oxidase (EC 1.4.3.6) activity was studied in regenerating rat liver after partial hepatectomy using inhibitors of protein and RNA syntheses. The administration to animals of cycloheximide or actinomycin D prevented the increase in diamine oxidase activity normally observed during the first hours after hepatectomy. The study of the turnover rate of diamine oxidase with cycloheximide demonstrated that the half-life of this enzyme was about 15 h in normal and regenerating liver. These results suggest that the rise in diamine oxidase activity in regenerating rat liver was due to the synthesis of new enzyme rather than to a lengthening of its turnover.     

The species distribution of xanthine oxidase

1. The distribution of xanthine oxidase in blood and tissues of various animals was studied by means of a radioactive assay capable of detecting 10(-7) unit of enzyme. The method was shown to be applicable to tissues with a high uricase content. 2. Of 16 mammalian species examined, six had low concentrations of xanthine oxidase in the serum. In six non-mammalian species, no activity was detected in the serum. 3. The enzyme was not found in the blood cells of any mammals, but was present in the nucleated red blood corpuscles of chicken, turtle and tortoise. 4. Studies of the tissue distribution in four species demonstrated high activities in the liver and intestinal mucosa and consistently low activities in skeletal muscle, heart and brain. 5. There is a rough correlation between the activity of enzyme in serum and its activity in lung tissue in 12 mammalian species. In the dog, left-atrial blood had higher concentrations of xanthine oxidase than right-atrial blood.     

Distribution of xanthine oxidoreductase activity in human tissues--a histochemical and biochemical study.

Localization of the activity of both the dehydrogenase and oxidase forms of xanthine oxidoreductase were studied in biopsy and postmortem specimens of various human tissues with a recently developed histochemical method using unfixed cryostat sections, poly-(vinyl alcohol) as tissue stabilizator, 1-methoxyphenazine methosulphate as intermediate electron acceptor and Tetranitro BT as final electron acceptor. High enzyme activity was found only in the liver and jejunum, whereas all the other organs studied showed no activity. In the liver, enzyme activity was found in sinusoidal cells and both in periportal and pericentral hepatocytes. In the jejunum, enterocytes and goblet cells, as well as the lamina propria beneath the basement membrane showed activity. The oxidase activity and total dehydrogenase and oxidase activity of xanthine oxidoreductase, as determined biochemically, were found in the liver and jejunum, but not in the kidney and spleen. This confirmed the histochemical results for these organs. Autolytic rat livers several hours after death were studied to exclude artefacts due to postmortem changes in the human material. These showed loss of activity both histochemically and biochemically. However, the percentage activity of xanthine oxidase did not change significantly in these livers compared with controls. The findings are discussed with respect to the possible function of the enzyme. Furthermore, the low conversion rate of xanthine dehydrogenase into xanthine oxidase during autolysis is discussed in relation to ischemia-reperfusion injury.     

Contamination of commercial preparations of xanthine oxidase by a Ca2+-dependent phospholipase A2

Using [1-14C]oleate-labelled autoclaved Escherichia coli as substrate, we demonstrate that many, but not all, commercial preparations of xanthine oxidase contain phospholipase A2 activity as a contaminant. Phospholipase A2 activity (64.3-545.6 nmol phospholipid hydrolyzed per min per mg protein) was optimal in the neutral to alkaline pH range, was Ca2+-dependent, and was unaffected by the addition of xanthine. Phospholipase A2 activity was totally inhibited by 1.0 mM EDTA while radical production by xanthine plus xanthine oxidase was unaffected by EDTA. Even chromatographically purified xanthine oxidase (Sigma Grade III) contained substantial phospholipase A2 activity (64.3 nmol/min per mg). Since the preparation of xanthine oxidase employs proteolytic digestion of milk or buttermilk by pancreatin, an extract of pancreas which is an organ rich in phospholipase A2 activity, we speculate that the contaminant phospholipase A2 is introduced by this treatment. Because xanthine oxidase is used extensively to study free radical-induced cell injury and membrane phospholipid alterations, the presence of a potent extracellular phospholipase A2 may have influenced previously published reports and such studies in the future should be interpreted with care.     

Activity of the glutathione antioxidant system and cytochrome P-450 in rat liver under induction and inhibition of xanthine oxidase

The effects of specific xanthine oxidase induction and inhibition on glutathione antioxidant system activity, lipid peroxidation, cytochrome P-450 quantity and corticosteroids concentration in the rat liver were studied. It was dependence established that there was a straight between xanthine oxidase activity and the activity of glutathione antioxidant system, lipid peroxidation and the ascorbic acid formation. The reciprocal dependence was established between xanthine oxidase activity and the concentrations of cytochrome P-450 and corticosteroids.     

Age-related changes in xanthine oxidase activity and lipid peroxidation,as well as in the correlation between both parameters,in plasma and several organs from female mice

Xanthine oxidase, a purine catabolism enzyme, has been implicated as an important source of oxidant production and plays an essential role in several inflammatory and oxidative stress-related diseases. It is known that the increasing levels of oxidants cause the chronic oxidative stress characteristic of the ageing process. The aim of the present work was to determine the changes in xanthine oxidase activity and oxidative damage to lipids in several organs (liver, kidney, spleen, lung and two different brain areas, namely cerebral cortex and brainstem) and plasma from two different age groups of BALB/c female mice: adult (7-month-old) and old (18-month-old) mice, as well as to analyse the possible correlation between both parameters. Xanthine oxidase activity was significantly increased in liver, cerebral cortex and plasma from old mice in comparison with adults. Similar results were obtained in the lipid peroxidation levels, in which old mice showed a high increment in liver and cerebral cortex. Moreover, the results show a significant and positive correlation between xanthine oxidase activity and lipid peroxidation levels in cerebral cortex. The age-related increase in the xanthine oxidase activity and lipid peroxidation in liver and cerebral cortex of mice seems to suggest that the xanthine oxidase plays a role in the acceleration of the oxidative damage in these organs with age and its possible contribution to the pathophysiological changes associated to the process of ageing.     

Sulfoxide reductase activity of liver aldehyde oxidase

The present study provides evidence that guinea pig and rabbit liver aldehyde oxidase (EC 1.2.3.1) in the presence of its electron donors such as aldehydes or N-heterocyclic compounds functions as a sulfoxide reductase towards sulindac and other sulfoxide compounds. In addition, the study shows that a combination of liver aldehyde oxidase and milk xanthine oxidase also exhibits sulfoxide reductase activity in the presence of xanthine, and electron donor of xanthine oxidase. Based on these facts, we propose a new electron-transfer system consisting of these two flavoenzymes.     

Influence of insulin status on extra-mitochondrial oxygen metabolism in the rat.

The effects of alloxan diabetes and subsequent treatment with insulin on extra-mitochondrial oxygen metabolism in terms of D-amino acid oxidase (DAAO), xanthine oxidase and catalase were examined. The DAAO activity in the liver with D-alanine and D-serine decreased by 33-62% in the diabetic group while the decrease in the kidneys was 61-74%. Insulin treatment resulted in overstimulation of DAAO activity in the liver but not in the kidneys. Tissue glycogen content was lowered in the diabetic animals but was restored by insulin treatment. Tissue glycogen content and DAAO activity showed an inverse relationship. The xanthine oxidase activity in the two tissues decreased from 40-55%; the catalase activity decreased from 34-54%. Insulin treatment was unable to restore the xanthine oxidase and catalase activities in both the tissues.     

Effect of tranquilizers of the 1,4-benzodiazepine series on the xanthine oxidase activity of the rat brain

Experiments in vivo and in vitro on 90 rats were made to study the influence of 1,4-benzodiazepine tranquilizers (phenazepam, nitrazepam and diazepam) on cerebral xanthine oxidase activity. Phenazepam, nitrazepam and diazepam in the dose of 5 mg per 200 g bw were shown to reduce xanthine oxidase activity by 80.4%, 64.3% and 55.8%, respectively 2 h after intraperitoneal injection. 6 h after the injection of benzodiazepines the enzyme activity grows, but control values are achieved only after nitrazepam injection. In vitro experiments revealed direct influence of the tranquilizers on xanthine oxidase. Phenazepam inhibits xanthine oxidase activity in concentration as long as 10(-10) M (to 36.6%), and practically completely in 10(-6) M concentration. Nitrazepam and diazepam inhibit xanthine oxidase activity within concentration range between 10(-8) M (to 51.5% and 33.2%, respectively), and 10(-4) M (practically completely). The inhibition of xanthine oxidase activity is shown to be caused by the competition between hypoxanthine, the reaction substrate, and tranquilizer, to bind with the active site of the enzyme.     

In vitro study of regulation of rat liver xanthine oxidase activity by antioxidant reducing agents]

It was learned the regulation of xanthine oxidase activity from rat liver in the partly purified prepared by ascorbic acid, glutathione-SH, dithiothreitol, cysteine++ and hydrocortisone++. It was shown that ascorbic acid glutathione-SH, dithiothreitol, and cysteine++ can be activators and uncompetitor inhibitors of xanthine oxidase in dependence from concentration. As far as hydrocortisone is concerned, it is a powerful uncompetitor inhibitor of xanthine oxidase, that is bind with it. It was considered the mechanism of activation and inhibition of xanthine oxidase by these reductors-antioxidants.     

A new spectrophotometric assay method of xanthine oxidase in crude tissue homogenate

A new spectrophotometric assay method of xanthine oxidase applicable to the crude tissue homogenate containing uricase was presented in this paper. By adding potassium 2,4-dihydroxy-6-carboxy-1,3,5-triazine (potassium oxonate) (0.1 mm) to the crude xanthine oxidase reaction system, uric acid was stoichiometrically formed from xanthine and detectable allantoin was not formed and the formation of uric acid was not influenced by uricase.Distribution of xanthine oxidase in various rat tissues was measured by this method, and it was shown that the activity was high in the liver, the small intestine, and the spleen. Uricase was shown to distribute mainly in the liver of rats.     

Molecular basis of the biological function of molybdenum. Developmental patterns of sulfite oxidase and xanthine oxidase in the rat

The developmental patterns of the molybdenum-containing enzymes sulfite oxidase and xanthine oxidase and of the mitochondrial enzymes adenylate kinase and succinate-cytochrome c reductase in rat liver are reported. Adenylate kinase and succinate-cytochrome c reductase develop in parallel with total liver protein and are maximal 5 days after birth. Sulfite oxidase, which is also a mitochondrial protein, shows its largest increase in activity between 5 and 11 days after birth. The appearance of sulfite oxidase and xanthine oxidase proteins parallels very closely the development of their respective activities. Xanthine oxidase activity is extremely low in rats prior to weaning at 21 days. Development of activity of this enzyme may be related to the protein nutritional status of the young animal. The development of both sulfite oxidase and xanthine oxidase activities is very much impaired by administration of tungsten to the pregnant rats for 20 days before birth of the litters. Apparently normal development of sulfite oxidase protein, however, leads to the accumulation of inactive molecules in the livers of offspring of tungsten-fed rats. Development of adenylate kinase and succinate-cytochrome c reductase activities is not affected by tungsten treatment.     

Electrophoretic analyses of alcohol dehydrogenase, aldehyde dehydrogenase, aldehyde oxidase, sorbitol dehydrogenase and xanthine oxidase from mouse tissues.

1. Cellulose acetate zymograms of alcohol dehydrogenase (ADH), aldehyde dehydrogenase, sorbitol dehydrogenase, aldehyde oxidase, "phenazine" oxidase and xanthine oxidase extracted from tissues of inbred mice were examined. 2. ADH isozymes were differentially distributed in mouse tissues: A2--liver, kidney, adrenals and intestine; B2--all tissues examined; C2--stomach, adrenals, epididymis, ovary, uterus, lung. 3. Two NAD+-specific aldehyde dehydrogenase isozymes were observed in liver and kidney and differentially distributed in other tissues. Alcohol dehydrogenase, aldehyde oxidase, "phenazine" oxidase and xanthine oxidase were also stained when aldehyde dehydrogenase was being examined. 4. Two aldehyde oxidase isozymes exhibited highest activities in liver. 5. "Phenazine oxidase" was widely distributed in mouse tissues whereas xanthine oxidase exhibited highest activity in intestine and liver extracts. 6. Genetic variants for ADH-C2 established its identity with a second form of sorbitol dehydrogenase observed in stomach and other tissues. The major sorbitol dehydrogenase was found in high activity in liver, kidney, pancreas and male reproductive tissues.