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PubMed1999

A tungsten supplemented diet attenuates bacterial translocation in chronic portal hypertensive and cholestatic rats: role of xanthine dehydrogenase and xanthine oxidase.

BACKGROUND: Bacterial translocation (BT) plays a major role in the pathophysiological process of spontaneous infections in portal hypertension (PH) and cholestatic jaundice. The major mechanisms promoting BT in experimental animal models are the disruption of the intestinal ecological equilibrium and disruption of the intestinal mucosal barrier. The enzymes xanthine dehydrogenase (XD) and xanthine oxidase (XO) are often implicated as a significant source of oxidants which have a major impact on the impairment of intestinal barrier function. AIM: To investigate the incidence of BT in rats with PH and obstructive jaundice, and to evaluate the impact of XD and XO. METHODS: Animals were subjected to sham laparotomy (SL), PH by calibrated stenosis of the portal vein, and common bile duct ligation (CBDL). They were fed either a standard pellet diet or a tungsten supplemented molybdenum-free diet. Four weeks after the operative procedure, intestinal colonisation and BT to portal vein, vena cava, mesenteric lymph nodes, liver, and spleen were determined. Intestinal XD and XO activity were measured enzymatically and histochemically. RESULTS: Significant (p<0.01) intestinal bacterial overgrowth was present in all PH and CBDL groups compared with the SL group. In normally fed animals after SL, BT occurred in 12%. In PH and after CBDL, the rate of BT increased significantly (p<0.05) to 28% and 54% respectively. In the jejunum of normally fed animals subjected to PH or CBDL, a significant increase in XO was observed (p<0.01). Animals fed a tungsten supplemented diet showed a significant attenuation of BT to 14% in PH and 22% after CBDL (p<0. 05). Tungsten treatment completely suppressed jejunal XD and XO activities. CONCLUSIONS: Significant intestinal bacterial overgrowth, BT, and XD to XO conversion occurred in PH and after CBDL. XD and XO inactivation by a tungsten supplemented molybdenum-free diet significantly reduced the incidence of BT without affecting intestinal bacterial overgrowth. These data strongly support the hypothesis that increased XD to XO conversion may contribute to intestinal barrier failure in PH and after CBDL.

Animals↗
PubMed1988

Mechanisms of conversion of xanthine dehydrogenase to xanthine oxidase in ischemic rat liver and kidney.

Previous studies have proposed and supported a role for the proteolytic, irreversible conversion of xanthine dehydrogenase to xanthine oxidase (XO) in postischemic injury in a wide variety of organs. A second mechanism of conversion, due to sulfhydryl modification and reversible with dithiothreitol (DTT), is potentially important but has not been well investigated. In this study rat liver and kidney were found to produce significant amounts of DTT-reversible XO during normothermic global ischemia. Formation of reversible XO precedes that of irreversible XO by approximately 0.5 h with a strong correlation (r = 0.92) existing between the rate of irreversible XO formation and the concentration of reversible XO. The formation of reversible XO is preceded by a depletion of glutathione with concentrations of glutathione during ischemia correlating (r = 0.85) with the observed concentration of reversible XO. While a large increase in the extent of liver damage occurs concurrently with conversion in an in vivo liver model of liver ischemia, an ischemia-reperfusion regimen (1 h of ischemia plus 0.5 h of reperfusion) that resulted in no conversion caused significant elevations in serum glutamic pyruvic transaminase and serum glutamic-oxaloacetic transaminase. Rats depleted of XO by tungsten dieting release 65% less enzyme after the same insult, suggesting that endogenous XO may also participate in the damage process independent of any conversion.

Animals↗
PubMed1996

Regulation of xanthine dehydrogenase and xanthine oxidase activity by hypoxia.

The present study determined the effect of hypoxia on xanthine dehydrogenase (XDH) and xanthine oxidase (XO) activity and gene and protein expression in cultured bovine aortic endothelial cells (BAEC). BAEC were exposed to hypoxia (3% O2) or anoxia (0% O2) for 24 or 48 h and to 24 h of hypoxia followed by 24 h of reoxygenation. Hypoxia- and anoxia-exposed BAEC demonstrated a greater than twofold increase in XDH/XO activity at 24 and 48 h compared with timed controls. Hypoxic cells that were subsequently reoxygenated in 21% O2 also demonstrated a similar increase in XDH/XO activity vs. timed controls. No differences were seen in mRNA levels at any time point. Similarly, no difference was noted in XDH/XO protein expression after hypoxic exposure, as determined by Western blot analysis. The increase in XDH/XO activity was not prevented by cyclohexamide, indicating that protein synthesis was not required. Thus the increased XDH/XO activity observed in response to hypoxia in the present study was due to posttranslational modulation of the enzyme.

Animals↗
PubMed1994

Nitric oxide inactivates xanthine dehydrogenase and xanthine oxidase in interferon-gamma-stimulated macrophages.

Interferon-gamma (IFN-gamma) has been reported to up-regulate transcription of the xanthine dehydrogenase (XDH) gene and to regulate XDH and xanthine oxidase (XO) activity in endothelial cells and liver tissue. Macrophages are a source of XDH/XO activity at inflammatory sites and are functionally regulated by IFN-gamma. We studied the effect of IFN-gamma on XDH and XO in rat bone marrow macrophages, rat alveolar macrophages, and murine RAW cells. Instead of an induction of enzyme activity, XDH/XO activity was almost totally lost after incubation with 100 to 1,000 U/ml of IFN-gamma for 24 h in all three cell types. The loss of cell-associated XDH/XO activity was not correlated with the appearance of XDH/XO activity in the media. In addition, the loss of XDH/XO activity could not be accounted for by transcriptional repression, since there was an increase in steady-state levels of XDH mRNA. To determine whether XDH/XO activity might be lost through nitric oxide-mediated inactivation of XDH/XO, we compared the time course and dose response for XDH/XO inactivation with that of nitric oxide production and found them similar. Treatment with the nitric oxide inhibitor N-monomethyl arginine appeared to totally block inactivation of XDH/XO by IFN-gamma. We conclude that upon stimulation with IFN-gamma, inducible nitric oxide in macrophages leads to post-transcriptional inhibition of XDH/XO, possibly minimizing the potential for tissue injury from XO released from macrophages into the inflammatory milieu. Inactivation of XDH may represent yet another "protective" role for nitric oxide at sites of inflammation.

Amino Acid Oxidoreductases↗
PubMed1992

Regulation of xanthine dehydrogenase and xanthine oxidase activity and gene expression in cultured rat pulmonary endothelial cells.

The central importance of xanthine dehydrogenase (XDH) and xanthine oxidase (XO) in the pathobiochemistry of a number of clinical disorders underscores the need for a comprehensive understanding of the regulation of their expression. This study was undertaken to examine the effects of cytokines on XDH/XO activity and gene expression in pulmonary endothelial cells. The results indicate that IFN-gamma is a potent inducer of XDH/XO activity in rat lung endothelial cells derived from both the microvasculature (LMVC) and the pulmonary artery. In contrast, interferon-alpha/beta, tumor necrosis factor-alpha, interleukin-1 or -6, lipopolysaccharide and phorbol myristate acetate have no demonstrable effect. The increase in XDH/XO activity requires new protein synthesis. By Northern analysis, IFN-gamma markedly increases the level of the 5.0-kb XDH/XO mRNA in LMVC. The increase is due, in part, to increased transcription rate of the XDH/XO gene. Transcriptional activation does not require new protein synthesis. The physiologic relevance of these observations was evaluated by administering IFN-gamma to rats. Intraperitoneal administration leads to an increased XDH/XO activity and XDH/XO mRNA level in rat lungs. In sum, IFN-gamma is a potent and biologically relevant inducer of XDH/XO expression; the major site of upregulation occurs at the transcriptional level.

Animals↗
PubMed2000

Study of the effect of the administration of Cd(II), cysteine, methionine, and Cd(II) together with cysteine or methionine on the conversion of xanthine dehydrogenase into xanthine oxidase.

Cadmium is known as to be a potent pulmonary carcinogen to human beings and to induce prostate tumor. The sequestration of cadmium, an extremely toxic element to living cells, which is performed by biological ligands such as amino acids, peptides, proteins or enzymes is important to minimize its participation in such deleterious processes. The synthesis of metallothionein is induced by a wide range of metals, in which cadmium is a particularly potent inducer. This protein is usually associated with cadmium exposure in man. Because metallothioneins may act as a detoxification agent for cadmium and chelation involves sulfur donor atoms, we administered only cadmium, cysteine, or methionine to rats and also each of these S-amino acids together with cadmium and measured the production of superoxide radicals derived from the conversion of xanthine dehydrogenase to xanthine oxidase. It could be seen in this work that the presence of cadmium enhances this conversion. However, its inoculation with cysteine or methionine almost completely diminishes this effect and this can be the result of the fact that these amino acids complex Cd(II). Thus, these compounds can be a model of the action of metallothionein, removing cadmium from circulation and preventing its deleterious effect.

Animals↗
PubMedDate not supplied

Xanthine oxidase and xanthine dehydrogenase from an estivating land snail.

During arousal from estivation in land snails, Otala lactea, active metabolic functions are restored within minutes and oxygen consumption increases dramatically. During the transition from the hypoxic conditions of estivation to normoxia it is possible that xanthine oxidase (XO) in hepatopancreas contributes to the observed lipid peroxidation. Using a fluorometric assay that is based on the oxidation of pterin, the activities and some properties of XO and XO+XDH (sum of XO and xanthine dehydrogenase activities) were measured in hepatopancreas extracts. Km values for pterin for XO and XO+XDH were 9 and 6 microM, respectively, and the Km of XDH for methylene blue was 5 microM. Both XO+XDH and XO activities were inhibited by allopurinol (I50 = 2 microM), pre-incubation at 40 degrees C, and by 5 min H2O2 pre-exposure. Inclusion of azide in the reaction promoted a rise of approximately 70-fold in the inactivation power of H2O2 due to inhibition of high endogenous catalase activity. The I50 for H2O2 of XO+XDH and XO activities in the presence of azide was 0.04 and 0.11 mM, respectively. Unlike the situation for mammalian XO, a previous reduction of O. lactea XO (by pterin) was not necessary to make the enzyme susceptible to H2O2 effects. Interestingly, methylene blue partially prevented both heat- and H2O2-induced inactivation of XO+XDH activity. These data indicate that the formation of an enzyme-methylene blue complex induces protection against heat and oxidative damage at the FAD-active site. Both XO and XO+XDH activites were significantly higher in snails after 35 days of estivation compared with active snails 24 h after arousal from dormancy. The ratio of XO/(XO+XDH) activities was also slightly increased in estivating O. lactea (from 0.07 to 0.09; P < 0.025). XO activity was 0.03 nmol.min-1.mg protein-1 in estivating snails. Compared with hepatopancreas catalase, XO activity is probably too low to contribute significantly to the net generation of oxyradicals, and hence to peroxidative damage. Rather, the low potential of XO to induce oxidative stress may constitute an adaptive advantage for O. lactea during arousal periods.

Acclimatization↗
PubMedDate not supplied

[Role of xanthine oxidase and xanthine dehydrogenase systems in thymocyte apoptosis, induced by papaverine].

It has been established that papaverine as well as other xenobiotics (dexamethasone and nitrosodimethylamine) [figure: see text] provoked the thymocyte death like apoptosis. The increase of the quantity of double-strand, single-strand DNA breaks and low molecular weight fragments of DNA preceded cell death. In papaverine-induced process of thymocyte apoptosis the total activity of xanthine oxidase in thymocytes strongly elevated long before their death, the conversion of xanthine dehydrogenase (D-form) to xanthinoxidase (O-form) and accumulation of O-form in the cultural medium took place. Direct stimulating effect of papaverine on O-form of enzyme in thymocyte lysate was revealed. The used digitonin thymocytes were divided into cytoplasmic and structural component fractions. It was shown that about 80% of total xanthinoxidase activity was concentrated in cytoplasma while only 20% of its activity was found in structural components. More higher ratio of xanthinoxidase/xanthindehydrogenase (XO/XDH) was observed and papaverine-induced changes of these enzyme forms activities were expressed more brightly in the structural components, than in the thymocyte cytoplasma. During the process of developing thymocytes apoptosis caused by papaverine the reaction of lipid peroxidation was intensified. XO-hypoxanthin system displaying prooxidant influence on cells increased the cytotoxic effect of papaverine but the presence of allopurinol or catalase and superoxidedismutase decreased it. Besides, cytotoxic action on thymocytes of allopurinol itself as well as hypoxanthin itself was revealed.

Animals↗
PubMed2003

Post-irradiation free radical generation: evidence from the conversion of xanthine dehydrogenase into xanthine oxidase.

The xanthine oxidoreductase (XOR) system which consists of xanthine dehydrogenase (XDH) and xathine oxidase (XO), is one of the major sources of free radicals in biological systems. The XOR system is pre-dominantly present as XDH in normal tissues and converts into the free radical generating XO-form in the damaged tissue. Therefore, the XO-form of the XOR system is expected to be mainly found in radiolytically damaged tissues. In such an event, XO may catalyze the generation of free radicals and potentiate radiation effects in the post-irradiation period. Recent findings on the effect of ionizing radiation on the XOR system in the liver of mice, peroxidative damage and lactate dehydrogenase support this possibility. From these results it has been hypothesized that free radical generating systems could be activated in the radiolytically damaged cell and in turn contribute to the cause and complications of late effects and their persistence in post-irradiation period. This aspect may have great significance in the understanding of radiation-induced damages. It may also have serious implication in various fields like radiation therapy, health physics, carcinogenesis, space travelling radiation exposures and post nuclear accident care. Further, it is suggested that efforts need to be made to search more system(s) which could be activated particularly at lower doses of radiation to generate free radicals in the post-exposure period.

Animals↗
PubMed1992

Effects of copper and selenium on electric parameters of cultured myocardial cells damaged by xanthine-xanthine oxidase.

Addition of xanthine 0.42 mmol.L-1 and xanthine oxidase 5.3 nmol.L-1 (X-XO) to the culture medium increased the amplitude of ESR spectra of myocardial cells, demonstrating an increase in free radical contents; diminished the action potential parameters significantly and reduced the input impedances from 0.34 +/- 0.11 to 0.24 +/- 0.1 M omega, expressing a typical electrical appearance of membrane damage. Supplying Cu 62.5 ng.ml-1 and/or Se 173 ng.ml-1 to the medium brought all of the electric parameters and the free radical content of myocardial cells back to normal. The results indicate that both the two trace elements are able to scavenge free radicals, thus antagonizing X-XO, which induces damage to myocardial cells.

Action Potentials↗
PubMed1994

Xanthine dehydrogenase and xanthine oxidase activity and gene expression in renal epithelial cells. Cytokine and steroid regulation.

Reactive oxygen species have been implicated in the tissue injury and loss of epithelial barrier function associated with a number of clinical disorders in which disregulated inflammation seems to be a dominant event, such as endotoxemia and viral syndromes. In these disorders, xanthine oxidase (XO) contained within the epithelial cell has been proposed as a major source of injurious reactive oxygen species. This study was undertaken in an effort to understand the regulation of xanthine dehydrogenase (XDH)/XO expression at both the activity and gene expression levels in the epithelial cell under conditions associated with the inflammatory response. The results indicate that TNF, IFN-gamma, IL-6, IL-1, and dexamethasone induce XDH/XO activity in bovine renal epithelial cells (MDBK). This pattern of XDH/XO regulation by cytokines and steroids is analogous to the profile of response seen by acute phase reactants. Metabolic labeling and immunoprecipitation revealed the increase in XDH/XO activity requires new protein synthesis. By Northern analysis, all cytokines and dexamethasone increased the level of the 5-kb XDH/XO mRNA. This increase was not detectable in the presence of actinomycin D but was further induced in the presence of cycloheximide, consistent with the major site of XDH/XO up-regulation occurring at the transcriptional level. XDH/XO mRNA was very stable, with no indication that the rates of transcript degradation contributed to differences in mRNA accumulation or ultimate activity levels. In addition to providing information on the regulation of XDH/XO, the data presented furthers the understanding of the epithelial cell's potential to actively respond to immunomodulators associated with injury/inflammation.

Animals↗
PubMed1993

The mechanism of conversion of xanthine dehydrogenase to xanthine oxidase in acute pancreatitis in the canine isolated pancreas preparation.

Four models of acute pancreatitis have been previously developed that use the ex vivo perfused isolated canine pancreas preparation. The four models include the intraarterial infusion of oleic acid (FFA) that mimics hyperlipemic pancreatitis, partial obstruction of the pancreatic duct with secretin stimulation (POSS) that mimics gallstone pancreatitis, a 2-hour period of ischemia before perfusion (ISCH 2) that mimics shock pancreatitis, and the infusion of cerulein at supramaximal stimulatory doses (CER), which lacks an obvious clinical counterpart. In the FFA, POSS, and ISCH 2 pancreatitis, but not in the CER pancreatitis, toxic oxygen metabolites, generated by the enzyme xanthine oxidase (XO), have been shown to be important mediators in the early pathogenesis. Ordinarily XO primarily occurs as xanthine dehydrogenase (XD) but can be converted to XO, which is the form that generates toxic oxygen metabolites. This conversion of XD to XO may take place either reversibly by way of sulfhydryl group oxidation or irreversibly by means of proteolytic cleavage of XD. This study was undertaken to investigate the mechanism of conversion of XD to XO in the FFA-, POSS-, and ISCH 2-induced pancreatitis models. CER pancreatitis was studied for comparison. After 4 hours of perfusion, pancreatitis was manifest by edema, weight gain, and hyperamylasemia in all four models. Dithiothreitol, a sulfhydryl group protector, ameliorated the weight gain in the FFA (40 +/- 14 gm to 18 +/- 13 gm; p < 0.05), POSS (28 +/- 10 gm to 9 +/- 3 gm; p < 0.05), and ISCH 2 pancreatitis (30 +/- 13 gm to 15 +/- 3 gm; p < 0.05), and ameliorated the hyperamylasemia in the POSS pancreatitis (12,062 +/- 4304 units/dl to 5877 +/- 2659 units/dl; p < 0.05). The CER pancreatitis was not ameliorated with dithiothreitol. A serine protease inhibitor of low molecular weight, phenylmethylsulfonyl fluoride, ameliorated only the CER pancreatitis (weight gain from 28 +/- 10 gm to 17 +/- 10 gm, p < 0.05; amylase activity from 38,116 +/- 6491 units/dl to 23,372 +/- 11,654 units/dl, p < 0.05), and not the FFA, POSS, or ISCH 2 pancreatitis. We conclude that in the three models of pancreatitis (FFA, POSS, and ISCH 2) that are mediated by toxic oxygen metabolites, XD is converted to XO reversibly by way of sulfhydryl group oxidation rather than irreversibly by way of proteolysis. In the CER pancreatitis, where XO does not play a role in the pathogenesis, proteolytic enzymes may be important mediators in the injury.

Acute Disease↗
PubMed1997

Spectrophotometric assay for superoxide dismutase based on tetrazolium salt 3'--1--(phenylamino)-carbonyl--3, 4-tetrazolium]-bis(4-methoxy-6-nitro)benzenesulfonic acid hydrate reduction by xanthine-xanthine oxidase.

XTT (3'-{1-[(phenylamino)-carbonyl]-3, 4-tetrazolium}bis(4-methoxy-6-nitro)benzenesulfonic acid hydrate) was reduced to a water-soluble product with an absorbance maximum at about 470 nm by superoxide anion generated by xanthine-xanthine oxidase (XO). The rate of XTT reduction was linearly related to XO activity and the reduction was inhibited by superoxide dismutase (SOD). A perfect inhibition of the reduction of XTT by SOD was achieved, suggesting that XTT does not interact with XO. The present XTT-based assay had a higher sensitivity than a conventional nitroblue tetrazolium-based assay by a factor of 2.5 at pH 10.2. This method was applicable to the SOD assay in the pH range 8.0-10.2.

Animals↗
PubMed1987

Action of xanthine-xanthine oxidase system on microsomal benzo(a)pyrene metabolism in vitro.

The effect of superoxide anion-radical and other reactive oxygen species on the metabolism of benzo(a)pyrene was studied with isolated mouse liver microsomes. Reactive oxygen species were generated in vitro by xanthine-xanthine oxidase plus Fe3+ X FeEDTA and benzo(a)pyrene metabolism was followed by reverse-phase high pressure liquid chromatography. The following results were obtained: The reactive oxygen species induced one-electron oxidation of benzo(a)pyrene and increased production of free epoxide as well as protein-binding intermediates. The reactive oxygen species triggered microsomal lipid peroxidation in the presence of Fe3+ X FeEDTA. As a result of microsomal lipid peroxidation a decreased activity of cytochrome P-450, epoxide hydrolase and UDP-glucuronyltransferase was found. It is suggested that active oxygen species changed the balance between bioactivation and conjugation of benzo(a)pyrene metabolites causing accumulation of the epoxide and protein-binding intermediates. The role of iron ions and chelates in this process is discussed.

Animals↗
PubMed1986

The xanthine derivative 1-(5'-oxohexyl)-3-methyl-7-propyl xanthine (HWA 285) enhances the actions of adenosine.

The effect of two closely related xanthine derivatives, pentoxifylline and HWA 285, on cyclic AMP accumulation in rat hippocampal slices and on adenosine uptake in erythrocytes was examined. Pentoxifylline was a weak competitive antagonist of adenosine effects on cyclic AMP accumulation. HWA 285, by contrast, had a small stimulatory effect per se and also potentiated the effect of adenosine (10-30 microM). Neither pentoxifylline nor HWA 285 significantly affected the cyclic AMP accumulation induced by the stable adenosine analogue NECA or by alpha- or beta-adrenoceptor activation. HWA 285 was a much more potent inhibitor of adenosine uptake into human erythrocytes than pentoxifylline and other examined xanthines including thiocaffeine, 8-p-sulphophenyltheophylline, theophylline, caffeine and enprofylline. It is suggested that HWA 285 may potentiate, rather than antagonize, the effects of endogenous as well as exogenous adenosine, partly as a consequence of adenosine uptake inhibition.

Adenosine↗
PubMedDate not supplied

[Researches on methyl-xanthine series.XI. Synthesis and physicochemical characterization of some 7-R-1,3-dimethyl-xanthine derivatives].

We have synthesized new xanthine compounds, derivatives of theophylline with various radicals in 7 position. The compounds have been characterized by molecular formula, weight, yield and melting point. The synthesized xanthine derivatives have been purified by repeated crystallizations from various organic solvents. Their chemical structure was confirmed by IR spectroscopy.

Bronchodilator Agents↗
PubMed1997

Alternations in beta-Adrenoceptor Mechanisms in Hearts Perfused With Xanthine Plus Xanthine Oxidase.

BACKGROUND: Although beta-adrenoceptors and adenylyl cyclase are known to be affected upon exposing cardiac membranes to some oxyradical generating systems, the results are conflicting. Furthermore, functional significance of alterations in the beta-adrenoceptor-adenylyl cyclase systems in terms of changes in the inotropic responses to catecholamines is not clear. METHODS AND RESULTS: The positive inotropic effect of isoproterenol was augmented on perfusing the isolated rat hearts with xanthine (X) plus xanthine oxidase (XO) for 5 minutes but was attenuated by perfusion for 15 minutes. The isoproterenol-stimulated adenylyl cyclase activity in cardiac membranes showed an increase at 10 minutes and a decrease at 30 minutes perfusion of hearts with X plus XO. The density of beta-adrenoceptors in cardiac membraners was reduced after 10 minutes and 30 minutes of perfusion with X plus XO, whereas the affinity of beta-adrenoceptors was increased after 10 minutes and reduced after 30 minutes. Although beta-adrenoceptors was increased after 10 minutes and reduced after 30 minutes. Although beta-adrenoceptors were unaltered by 10 minutes of perfusion with X plus XO, their affinity was increased and density was decreased by 30 minutes of perfusion. The agonist competition curves using isoproterenol indicated an increase in the number of coupled receptors in the high affinity state on 10 minutes of perfusion and an increase in the low affinity state of coupled receptor due to 30 minutes of perfusion with X plus XO. The basal as well as forskolin-, NaF- and Gpp(NH)p-stimulated adenylyl cyclase activities in cardiac membranes exhibited an increase after 10 minutes and decrease after 30 minutes of perfusion with X plus XO. Although the presence of superoxide dismutase plus catalase in the perfusion medium prevented most of the alterations due to X plus XO, it did not alter the increased affinity of the beta-adrenoceptor upon perfusing hearts for 10 minutes with X plus XO. CONCLUSIONS: The results in this study suggest the biphasic nature of the oxyradical-induced alterations in both the inotropic responses to catecholamines and the beta-adrenoceptor-mediated signal transduction mechanism in the heart.

Journal Article↗
PubMed1996

Purification of rat liver xanthine oxidase and xanthine dehydrogenase by affinity chromatography on benzamidine-sepharose.

The oxidase form of xanthine dehydrogenase (XO; EC 1.1.3.22) has been purified approximately 200-fold from rat liver extracts using a three-step process of heat treatment, ammonium sulfate precipitation, and chromatography on benzamidine-Sepharose. The purified enzyme showed only minor contamination when analyzed by gel electrophoresis under either native or sodium dodecyl sulfate (SDS)-denatured conditions and appears to be intact based on its subunit size on SDS-polyacrylamide gel electrophoresis, its N-terminal amino acid sequence, and its ability to be converted to the NAD-dependent dehydrogenase form (XD; EC 1.1.1.204) by incubation with dithiothreitol. Isoelectric focusing analysis showed that the purified enzyme consists of two major, enzymatically active isoforms with average pI values of 6.13 and 6.23 and a minor enzymatically active isoform with an average pl value of 6.07. A similar purification of XD was achieved by preincubating the partially purified oxidase with dithiothreitol prior to affinity chromatography on benzamidine-Sepharose. The effects of benzamidine on the kinetic properties of purified rat XO were characterized at pH 8 and 9 and were compared to those of bovine milk XO. Benzamidine was found to be a weak competitive inhibitor of the purified rat enzyme with Ki values of 30 and 10 mM at pH 8 and 9, respectively. In contrast, the Ki values for benzamidine with bovine XO were more than 10-fold greater. The findings presented in this study show that benzamidine is a competitive inhibitor of XO and that affinity chromatography on benzamidine-Sepharose provides a simple, rapid, and effective means of purifying both the oxidase and dehydrogenase forms of rat XO.

Aldehyde Oxidase↗