Optical and electron paramagnetic resonance spectroscopic studies on purine hydroxylase II from Aspergillus nidulans

Purine hydroxylase II from Aspergillus nidulans contains a molybdenum cofactor very similar to that found in a number of other molybdenum-containing hydroxylases. (A. nidulans contains two purine hydroxylases, I and II, related to each other by possession of a common cofactor and overlapping substrate specificity.) Addition of reducing substrates effects bleaching of the visible absorption spectrum of the enzyme, the decrease in absorbance at 450 nm being linearly proportional to that at 550 nm. No increase in absorption at longer wavelengths was observed during such titrations. Electron paramagnetic resonance studies of reduced samples of native and modified enzyme species showed the presence of a number of Mo(V) signals (gav = 1.97), exhibiting H hyperfine coupling, comparable to those in the corresponding enzymes from other sources. The enzyme possesses two non-heme-iron-sulfur centers, one (Fe2S2)I with gav less than 2.0 and the other (Fe2S2)II with gav greater than 2.0. The flavin radical signal observed at pH 7.8 had a linewidth of 1.5 mT, indicating it to be the anionic form FAD- . In this respect purine hydroxylase II is unique among all molybdenum-containing hydroxylases studied to date.     

Purpurogallin is a more powerful protector of kidney cells than Trolox and allopurinol.

Phase contrast and electron microscopic experiments demonstrated that oxyradicals generated with xanthine oxidase and hypoxanthine markedly damage rat kidney mesangial and porcine tubular epithelial cells. Purpurogallin, a phenol found in oak nutgalls, prolongs survival of the xanthine oxidase exposed renal cells three- to nine-fold longer than those without purpurogallin present. At levels equimolar to purpurogallin, either Trolox or allopurinol is less effective in delaying cell necrosis. Purpurogallin scavenges not only xanthine oxidase generated oxyradicals, but also non-enzymatically produced peroxyl radicals, more actively than equimolar levels of Trolox or allopurinol. Purpurogallin inhibits xanthine oxidase with severalfold higher potency than allopurinol and its more active metabolite oxypurinol. Therefore, purpurogallin is a stronger antioxidant than Trolox and a more potent inhibitor of xanthine oxidase than allopurinol as well as oxypurinol.     

Inhibition of nodule functioning in cowpea by a xanthine oxidoreductase inhibitor, allopurinol

Allopurinol (1H-pyrazolo-[3,4-d]pyrimidine-4-ol), an inhibitor of xanthine oxidation in ureide-producing nodulated legumes, was taken up from the rooting medium, translocated in xylem, and transferred to nodules of both the ureide-forming cowpea (Vigna unguiculata L. Walp.) and the amide-forming white lupin (Lupinus albus L.). Cowpea suffered severe nitrogen deficiency, extreme chlorosis, and reduced growth, whereas lupin was unaffected by the inhibitor. Similar results were obtained with oxypurinol (1H-pyrazolo-[3,4-d]pyrimidine-4,6-diol). Xylem composition of symbiotic cowpea was markedly changed by allopurinol. Ureides fell to a very low level, but xanthine and, to a lesser extent, hypoxanthine increased markedly. Xylem glutamine was also reduced, but there was little change in other amino acids. Nitrogenase (EC 1.7.99.2) activity of intact nodulated plants or nodulated root segments of plants treated with allopurinol or oxypurinol for 24 hours or more was severely inhibited in cowpea but unaffected in lupin for periods of exposure up to 9 days. Nitrogenase activity of slices of nodules prepared from allopurinol-treated cowpea showed inhibition comparable to that of intact plants. Breis prepared from nodules of treated plants showed no reduction in nitrogenase, nor was there reduction in activity of breis following addition of allopurinol, xanthine, or a range of purine pathway intermediates. Increasing the O₂ concentration in assays above 20% (volume/volume) reversed inhibition of nitrogenase by allopurinol in intact nodulated roots. It was concluded for cowpea that allopurinol not only inhibited ureide synthesis but also caused inhibition of nitrogenase activity, thereby leading to progressive dysfunction and eventual senescence of nodules. The mechanistic relationships between inhibition of ureide biosynthesis, changes in gaseous diffusion resistance, and reduced nitrogenase activity remain obscure.     

Selectivity of febuxostat, a novel non-purine inhibitor of xanthine oxidase/xanthine dehydrogenase

The purine analogue, allopurinol, has been in clinical use for more than 30 years as an inhibitor of xanthine oxidase (XO) in the treatment of hyperuricemia and gout. As consequences of structural similarities to purine compounds, however, allopurinol, its major active product, oxypurinol, and their respective metabolites inhibit other enzymes involved in purine and pyrimidine metabolism. Febuxostat (TEI-6720, TMX-67) is a potent, non-purine inhibitor of XO, currently under clinical evaluation for the treatment of hyperuricemia and gout. In this study, we investigated the effects of febuxostat on several enzymes in purine and pyrimidine metabolism and characterized the mechanism of febuxostat inhibition of XO activity. Febuxostat displayed potent mixed-type inhibition of the activity of purified bovine milk XO, with Ki and Ki' values of 0.6 and 3.1 nM respectively, indicating inhibition of both the oxidized and reduced forms of XO. In contrast, at concentrations up to 100 muM, febuxostat had no significant effects on the activities of the following enzymes of purine and pyrimidine metabolism: guanine deaminase, hypoxanthine-guanine phosphoribosyltransferase, purine nucleoside phosphorylase, orotate phosphoribosyltransferase and orotidine-5'-monophosphate decarboxylase. These results demonstrate that febuxostat is a potent non-purine, selective inhibitor of XO, and could be useful for the treatment of hyperuricemia and gout.     

Inhibition of mammalian xanthine oxidase by folate compounds and amethopterin

We have examined the effects of folate compounds and the folate analog amethopterin (methotrexate) as inhibitors of mammalian xanthine oxidase and have found that they offer potent inhibition of the enzyme. We have compared the inhibitory potency of folic acid and its coenzyme derivative tetrahydrofolic acid to that of allopurinol, a known inhibitor of xanthine oxidase, and have demonstrated that folic acid and tetrahydrofolic acid are severalfold more potent than allopurinol as inhibitors of xanthine oxidase. Comparative inhibition constants calculated were 5.0 X 10(-7) M for folic acid. 1.25 X 10(-6) M for tetrahydrofolic acid, and 4.88 X 10(-6) M for allopurinol. Incubation of xanthine oxidase with folic acid at a concentration of 10(-6) M abolished 94% of the enzymic activity within 1 min of incubation with the enzyme. At the same concentration, allopurinol was almost ineffective as an inhibitor of xanthine oxidase. The substrate xanthine protected the enzyme against total inhibition by folic acid. Reversibility of the enzymic inhibition by folic acid was demonstrated. Folic acid-inactivated enzyme was totally regenerated either by filtration through Sephadex G-200 or by precipitation with ammonium sulfate. 2-Amino-4-hydroxypteridine was a poor substrate for the enzyme but a potent inhibitor for the oxidation of xanthine by the enzyme. The inhibition constant calculated was 1.50 X 10(-6) M. In the presence of an excess of xanthine oxidase, neither folic acid nor tetrahydrofolic acid and allopurinol exhibited any change in intensity of their absorbance or in the wavelength of their maximal absorbance that might have been suggestive of substrate utility. The folate analog amethopterin was also determined a potent inhibitor of mammalian xanthine oxidase. The inhibition constant calculated was 3.0 X 10(-5) M.     

Purine Catabolism in Advanced Carotid Artery Plaque

This study was carried out on carotid artery plaque and plasma of 50 patients. We analyzed uric acid, hypoxanthine, xanthine, and allantoin levels to verify if enzymatic purine degradation occurs in advanced carotid plaque; we also determined free radicals and sulphydryl groups to check if there is a correlation between oxidant status and purine catabolism. Comparing plaque and plasma we found higher levels of free radicals, hypoxanthine, xanthine, and a decrease of some oxidant protectors, such as sulphydryl groups and uric acid, in plaque. We also observed a very important phenomenon in plaque, the presence of allantoin due to chemical oxidation of uric acid, since humans do not have the enzyme uricase. The hypothetical elevated activity of xanthine oxidase in atherosclerosis could be reduced by specific therapies using its inhibitors, such as oxypurinol or allopurinol.     

Purine catabolism in advanced carotid artery plaque

This study was carried out on carotid artery plaque and plasma of 50 patients. We analyzed uric acid, hypoxanthine, xanthine, and allantoin levels to verify if enzymatic purine degradation occurs in advanced carotid plaque; we also determined free radicals and sulphydryl groups to check if there is a correlation between oxidant status and purine catabolism. Comparing plaque and plasma we found higher levels of free radicals, hypoxanthine, xanthine, and a decrease of some oxidant protectors, such as sulphydryl groups and uric acid, in plaque. We also observed a very important phenomenon in plaque, the presence of allantoin due to chemical oxidation of uric acid, since humans do not have the enzyme uricase. The hypothetical elevated activity of xanthine oxidase in atherosclerosis could be reduced by specific therapies using its inhibitors, such as oxypurinol or allopurinol.     

On the Specificity of Allopurinol and Oxypurinol as Inhibitors of Xanthine Oxidase. A Pulse Radiolysis Determination of Rate Constants for Reaction of Allopurinol and Oxypurinol with Hydroxyl Radicals

Allopurinol has been employed as a “specific” inhihitor of xanthine oxidase in studies of hypoxic/ reoxygenation injury. Pulse radiolysis was used to establish rate constants for the reactions of allopurinol and its major metabolite oxypurinol with hydroxyl radicals: values were (1.45 ± 0.241 × 10⁹ M^-1 s^-1 for allopurinol and (4.95 ± 0.84) × 10⁹ M^-1 s^-1 for oxypurinol. These rate constants show that, in view of the amounts of allopurinol that have been used in animal studies. hydroxyl radical scavenging by this molecule could contribute to its biological actions. especially if animals are pre-treated with allopurinol. so allowing oxypurinol to form. The ability of allopurinol to protect tissues not containing xanthine oxidase against reoxygenation injury may be related to radical scavenging by allopurinol and oxypurinol.     

On the Specificity of Allopurinol and Oxypurinol as Inhibitors of Xanthine Oxidase. A Pulse Radiolysis Determination of Rate Constants for Reaction of Allopurinol and Oxypurinol with Hydroxyl Radicals

Allopurinol has been employed as a “specific” inhihitor of xanthine oxidase in studies of hypoxic/ reoxygenation injury. Pulse radiolysis was used to establish rate constants for the reactions of allopurinol and its major metabolite oxypurinol with hydroxyl radicals: values were (1.45 ± 0.241 × 10⁹ M^-1 s^-1 for allopurinol and (4.95 ± 0.84) × 10⁹ M^-1 s^-1 for oxypurinol. These rate constants show that, in view of the amounts of allopurinol that have been used in animal studies. hydroxyl radical scavenging by this molecule could contribute to its biological actions. especially if animals are pre-treated with allopurinol. so allowing oxypurinol to form. The ability of allopurinol to protect tissues not containing xanthine oxidase against reoxygenation injury may be related to radical scavenging by allopurinol and oxypurinol.     

Unusual hypoxanthine hydroxylation system in hepatopancreas of Helix pomatia (Gastropoda)

The enzymatic system in hepatopancreas of H. pomatia (terrestrial purinotelic gastropod) hydroxylates hypoxanthine to xanthine and uric acid but fails to hydroxylate adenine, nicotinic acid and 3-methyl-6- hydroxypurine ; allopurinol is hydroxylated to oxypurinol 7 times faster than hypoxanthine to xanthine; at concentration of 10(-6) M it inhibits hydroxylation of hypoxanthine by 55%. Two protein fractions [precipitated at 0-0.30 (I) and 0.30-0.45 (II) saturation with (NH4)2 SO4] hydroxylate hypoxanthine with NAD+ as a cosubstrate but only fraction I, predominating during the active life, hydroxylates also xanthine and is inhibited by NADH. Protein fraction II, dominant during winter sleep, does not hydroxylate xanthine and its hypoxanthine-hydroxylating activity is not inhibited by NADH. The latter property may enable continuous operation of the protein catabolic pathway under anaerobiosis.     

6-(N-benzoylamino)purine as a novel and potent inhibitor of xanthine oxidase: inhibition mechanism and molecular modeling studies

The inhibition of xanthine oxidase (XO) activity by the purine analogue 6-(N-benzoylamino)purine was evaluated and compared with the standard inhibitor, allopurinol and the parent compound adenine. 6-(N-benzoylamino)purine is a highly potent inhibitor of XO (IC50 = 0.45 microM) and comparable to allopurinol (IC50 = 0.80 microM). Furthermore, 6-(N-benzoylamino)purine neither produced any enzymatic superoxide nor reduced XO by an electron transfer reaction unlike allopurinol. 6-(N-benzoylamino)purine (Ki = 0.0475 microM) is about 10000-fold more potent as a XO inhibitor compared to the only known purine analogue 8-bromoxanthine (Ki = 400 microM). 6-(N-Benzoylamino)purine is a competitive inhibitor of XO and the inhibition was not completely reversed even at 100 microM xanthine concentration. The calculated interaction energy [Ecomplex - (Eligand + Eprotein)] of -30.5, -22.6, and -17.2 kcal/mol, respectively, of 6-(N-benzoylamino)purine, 8-bromoxanthine and the parent compound adenine provided the rationale for the better enzyme inhibitory activity of 6-(N-benzoylamino)purine. To understand the role of the benzamido group in the inhibition process, molecular docking studies were carried out and it was revealed that the hydrogen bonding interactions involving N-7 of the purine ring and the N-H of Arg880, N-H of the purine ring and OH of Thr1010, as well as non-bonded interactions of the benzamido group of 6-(N-benzoylamino)purine with amino acid residues Gly799, Glu802, Phe914, Ala1078, Ala1079 and Glu1261 in the active site of XO play an important role in the stabilization of the E-I complex.     

A mutation defective in the xanthine alternative pathway of Aspergillus nidulans: its use to investigate the specificity of uaY mediated induction.

Summary In Aspergillus nidulans uric acid can be produced from xanthine via purine hydroxylase I (xanthine dehydrogenase) or via the xanthine alternative pathway (Darlington and Scazzocchio, Biochem. Biophys. Acta, 166, 569–571; 1968). A mutation defective in the xanthine alternative pathway of Aspergillus nidulans is described. By combining this mutation with hxB-20 which results in complete loss of purine hydroxylase I and II activities, but which conserves cross-reacting material, it is possible to block completely uric acid production and thus investigate which are the effective in vivo inducers of three enzymes under the control of the positive regulatory gene uaY: adenine deaminase, purine hydroxylase I (measured as cross-reacting material) and urate oxidase. It is concluded that uric acid is the only effective physiological inducer, while its 2 and 8 thio-analogues serve as gratuitous inducers.     

Reaction of fluorescein bimercuric acetate with a molybdenum center of xanthine oxidase from milk

Inhibition of milk xanthine oxidase by fluorescein bimercuriacetate (FMA) allows for the classification of S-containing groups according to their localization and role in the catalytic activity of the enzyme. The enzyme (E) complexes with FMA (E--FMA I and E--FMA II) differing in their activity, stoichiometry and spectral properties were studied at various experimental conditions, reaction time and FMA concentrations. The enzyme molecule contains 5 groups that are reactive towards FMA (E--FMA I) and are localized outside the active center. That these groups have no concern with activity and are subjected to modification irrespective of whether or not the xanthine oxidase molecule has an intact Mo-center. The formation of an inactive E--FMA II complex is associated with an additional (in comparison with E--FMA I) binding of two FMA molecules per molecule of the active enzyme. The stoichiometry of the E--FMA II complex was determined by the X-ray fluorescent method from the amount of the Hg in enzyme. A kinetic scheme of xanthine oxidase inhibition by FMA is proposed, according to which the inhibition is a result of modification of two groups in the enzyme active center, of which only one is essential for the enzyme activity. This scheme also postulates the role of reversible E--FMA complexes in the course of irreversible inhibition. Xanthine oxidase is protected against FMA by the substrate (xanthine), competitive inhibitors (azaxanthine and allopurinol) and acceptor (2,6-dichlorophenolindophenol), i. e., compounds which interact with the Mo-center of the enzyme. The EPR spectra of the dithionite-reduced E--FMA II complex were found to contain a "slow" signal, Mo(V), typical of the Mo-center devoid of labile sulphur. It was assumed that the essential group interacting with FMA in the active center of xanthine oxidase as a terminal sulphur which is a component of the coordination region of Mo.     

Xanthine oxidase and adenosine deaminase in commercial bovine spleen phosphodiesterase preparations.

Commercial bovine spleen phosphodiesterase preparations contain xanthine oxidase activity; the xanthine oxidase in such preparations mediates the oxidation of a pteridine derivative as well as a standard purine substrate (hypoxanthine). The xanthine oxidase activity in the phosphodiesterase preparations is inhibited strongly by allopurinol (4-hydroxypyrazolo(3,4-d) pyrimidine). The reported ability of phosphodiesterase preparations to catalyze the deamination of adenosine derivatives appears to be due to contamination with a conventional adenosine deaminase in view of the observations that this activity is inhibited by an established inhibitor of adenosine deaminase and that the relative rates of deamination of N¹-methyladenosine and adenosine are similar with both the phosphodiesterase preparation and calf intestine adenosine deaminase.     

Hyperthermia enhances the cytotoxic effects of reactive oxygen species to Chinese hamster cells and bovine endothelial cells in vitro.

Hyperthermia is under intensive investigation as a treatment for tumors both alone and in combination with other therapeutic agents. Hyperthermia has a profound effect on the function and structural integrity of tumor microvasculature; this has often been cited as a reason for its effectiveness in treatment of tumors. To test the role of hyperthermia in cytotoxic effects of active oxygen species, Chinese hamster, V79, and bovine endothelial cells were treated by the active oxygens, O not equal to 2 and H2O2, generated from the hypoxanthine/purine and xanthine oxidase reactions. It was found that cytotoxicity to V79 cells depends on the concentrations of purine and xanthine oxidase. A high level of cytotoxicity may be initiated in hyperthermia-treated tumors because high xanthine oxidase activity is known to be associated with tumors and endothelial cells, and degradation processes produce high concentrations of xanthine oxidase substrates in tumors. Since the cytotoxic effect can be reduced by the xanthine oxidase inhibitor, allopurinol, and the H2O2 removal enzyme, catalase, the cytotoxic effect in this experimental system is dependent on xanthine oxidase and H2O2. Adding erythrocytes at the same time as purine and xanthine oxidase could also prevent the cytotoxicity. Elevated temperatures stimulated the reaction of purine and xanthine oxidase and resulted in an increased cytotoxic effect. A similar effect is observed in growth inhibition and colony formation in endothelial cells without adding xanthine oxidase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Analysis of purines in urinary calculi by high-performance liquid chromatography

A high-pressure liquid chromatography method has been developed for the analysis in urinary calculi of six purines: uric acid, 2, 8-dihydroxyadenine, xanthine, hypoxanthine, allopurinol, and oxypurinol. Separation was conducted isocratically on a reversed-phase column, using 50 mM phosphate buffer (pH 5.5) / methanol (97/3, v/v) as mobile phase. Limits of detection, depending on compound, ranged from 7 to 28 microg/g stone weight. Hitherto, no reports have appeared on other purines present with uric acid in stones, due to lack of a sensitive and specific analytical method. We have now found that all calculi with more than 4% uric acid also contained 1-methyluric and 7-methyluric acids and trace amounts of hypoxanthine, xanthine, and 2,8-dihydroxyadenine. Accurate identification and quantitation of purines in urinary calculi are important for the diagnosis of rare metabolic diseases leading to urolithiasis (xanthinuria, dihydroxyadeninuria), as well as for prevention of iatrogenic complications during treatment with allopurinol of uric acid urolithiasis. The method may be used for reference purposes in clinical laboratories and for research on the pathogenesis of urolithiasis in disorders of purine metabolism.     

Coordinate regulation of purine nucleoside phosphorylase and xanthine dehydrogenase levels in chick liver

Previously it has been shown that the levels of xanthine dehydrogenase in chick liver can be increased by feeding high-protein diets, adenine, and allopurinol (a xanthine dehydrogenase inhibitor). Also, it has been shown that starvation increases the level of xanthine dehydrogenase in chick liver and that unsaturated fatty acids in the diet suppress the levels of xanthine dehydrogenase in the liver. Results reported here show that starvation and high-protein diets enhance the levels of purine nucleoside phosphorylase and that unsaturated fatty acids suppress the level of this enzyme. In contrast with xanthine dehydrogenase, adenine and allopurinol have no effect on purine nucleoside phosphorylase levels. These results suggest that dietary protein and unsaturated fatty acids regulate more than one enzyme involved in the production of uric acid.Levels of xanthine dehydrogenase in the pancreas can be increased by feeding and decreased by starvation or feeding unsaturated fatty acids. None of these procedures has any effect on the level of pancreatic purine nucleoside phosphorylase.     

Alopurinol y su papel en el tratamiento de la sarcopenia

Xanthine oxidase (XO) is an enzyme that catalyzes the oxidation of hypoxanthine to xanthine and uric acid and plays an important role in purine catabolism. The purine analogue, allopurinol, is a well-known inhibitor of XO widely used in the clinical management of gout and conditions associated with hyperuricemia. More recent data indicate that allopurinol reduces oxidative stress and improves vascular function in several cardiometabolic diseases, prolongs exercise time in angina, and improves the efficiency of cardiac contractility in heart failure. XO also plays an important role in free radical generation during skeletal muscle contraction and thus, it has been related to the muscle damage associated to exhaustive exercise. Several research groups have shown the protective effect of allopurinol in the prevention of this type of damage.     

Propranolol as xanthine oxidase inhibitor: implications for antioxidant activity

Propranolol is the beta-blocker most widely used in the management of cardiovascular disorders. It has been proposed that propranolol may act as a "chain-breaking" antioxidant. We have directly examined the ability of propranolol to inhibit superoxide-dependent, iron-promoted cardiac membrane phospholipid peroxidation, with xanthine oxidase (XOD) as a physiologically-recognized, enzymatic superoxide generator. Our results demonstrate that propranolol not only protects cardiac-membrane lipid from peroxidative damage, but also acts as a simple, reversible XOD inhibitor, noncompetitive with xanthine substrate. Propranolol, at effective antiperoxidant and XOD-inhibitory concentrations, cannot scavenge superoxide radical. The antiperoxidative profile of propranolol resembles that of the known XOD inhibitor allopurinol, although allopurinol, a tight-binding substrate-analog competitive with xanthine, inhibits XOD in a manner mechanistically very different from that of propranolol. Furthermore, the antiperoxidative profiles of both propranolol and allopurinol do not resemble those of chain-breaking antioxidants such as alpha-tocopherol. These data, along with the tendency of propranolol to concentrate in myocardial membranes and cytosol, suggest that the observed antioxidant action of propranolol, as a consequence of XOD inhibition, could play a pharmacologic role in propranolol's cardioprotective effects.     

Simultaneous determination of 16 purine derivatives in urinary calculi by gradient reversed-phase high-performance liquid chromatography with UV detection

A reversed-phase high-performance liquid chromatography (HPLC) method with ultraviolet detection has been developed for the analysis of purines in urinary calculi. The method using gradient of methanol concentration and pH was able to separate 16 compounds: uric acid, 2,8-dihydroxyadenine, xanthine, hypoxanthine, allopurinol and oxypurinol as well as 10 methyl derivatives of uric acid or xanthine (1-, 3-, 7- and 9-methyluric acid, 1,3-, 1,7- and 3,7-dimethyluric acid, 1-, 3- and 7-methylxanthine). Limits of detection for individual compounds ranged from 0.006 to 0.035 mg purine/g of the stone weight and precision (CV%) was 0.5-2.4%. The method enabled us to detect in human uric acid stones admixtures of nine other purine derivatives: natural metabolites (hypoxanthine, xanthine, 2,8-dihydroxyadenine) and methylated purines (1-, 3- and 7-methyluric acid, 1,3-dimethyluric acid, 3- and 7-methylxanthine) originating from the metabolism of methylxanthines (caffeine, theophylline and theobromine). The method allows simultaneous quantitation of all known purine constituents of urinary stones, including methylated purines, and may be used as a reference one for diagnosing disorders of purine metabolism and research on the pathogenesis of urolithiasis.