Development of the pteridine pathway in the zebrafish, Danio rerio

In the zebrafish, the peripheral neurons and the pigment cells are derived from the neural crest and share the pteridine pathway, which leads either to the cofactor tetrahydrobiopterin or to xanthophore pigments. The components of the pteridine pattern were identified as tetrahydrobiopterin, sepiapterin, 7-oxobiopterin, isoxanthopterin, and 2,4,7-trioxopteridine. The expression of GTP cyclohydrolase I activity during the first 24-h postfertilization, followed by 6-pyruvoyl-5,6,7,8-tetrahydropterin synthase and sepiapterin reductase, suggest an early supply of tetrahydrobiopterin for neurotransmitter synthesis in the neurons and for tyrosine supply in the melanophores. At 48-h postfertilization, sepiapterin formation branches off the de novo pathway of tetrahydrobiopterin synthesis. Sepiapterin, via 7,8-dihydrobiopterin and biopterin, serves as a precursor for the formation of 7-oxobiopterin, which may be further catabolized to isoxanthopterin and 2,4,7-trioxopteridine. Neither 7, 8-dihydrobiopterin nor biopterin is a substrate for xanthine oxidoreductase. In contrast, both of these compounds are oxidized at C-7 by a xanthine oxidase variant form, which is inactivated by KCN, but is insensitive to allopurinol. The oxidase and the dehydrogenase form of xanthine oxidoreductase as well as the xanthine oxidase variant have specific developmental patterns. It follows that GTP cyclohydrolase I, the formation of sepiapterin, and the xanthine oxidoreductase family control the pteridine pathway in the zebrafish.     

The effect of nutritional state and allopurinol on nucleotide formation in enterocytes from the guinea pig small intestine

The uptake of purine nucleosides (guanosine and hypoxanthine) and bases (guanine, hypoxanthine and adenine) and their incorporation into nucleotides were studied in enterocytes isolated from fed and 3-day fasted guinea pig jejunum. Both total uptake and synthesis of nucleotides were greater for these purines in the fasted, as compared to the fed state for the first 5 min, when the initial substrate concentration in the medium was 10 microM. Increased uptake did not result from a change in the relative distribution of synthesized nucleotides between the fed and fasted states. Reduced catabolism was observed in the medium by enterocytes from fasted as compared to fed animals after 1 min of incubation with both inosine and guanosine. Preincubation of enterocytes with allopurinol (a xanthine oxidase inhibitor) decreased total uptake but increased the formation of IMP from hypoxanthine. Xanthine oxidase activity measured in mucosa from fasted guinea pigs was lower than that from fed animals (6.29 vs. 9.30 nmol/min per mg protein, respectively). However, activities of the salvage enzymes adenine phosphoribosyltransferase and hypoxanthine-guanine phosphoribosyltransferase were not significantly different between the fed and fasted states. These data show that allopurinol treatment, and mucosal atrophy resulting from fasting, decrease xanthine oxidase activity and increase nucleotide synthesis from exogenous substrates in enterocytes from the guinea-pig small intestine, suggesting a regulatory function of mucosal xanthine oxidase in purine salvage by the small intestine.     

EPR studies on the superoxide-scavenging capacity of the nutraceutical resveratrol

Resveratrol (3,4',5-trihydroxystilbene), a polyphenolic compound found in mulberries, grapes, and red wine, has received considerable attention because of its apparent protective effects against various degenerative diseases due to its potential antioxidant activities. However, direct evidence for the superoxide-scavenging capacity of resveratrol is lacking in literature. In this study, electron paramagnetic resonance spectroscopy in combination with 5-(diethoxyphosphoryl)-5-methylpyrroline-N-oxide (DEPMPO)-spin trapping technique was utilized to determine the ability of resveratrol in scavenging superoxide anions generated from both potassium superoxide and the xanthine oxidase/xanthine system. We have demonstrated here for the first time that the presence of resveratrol resulted in decreased formation of DEPMPO-superoxide adduct (DEPMPO-OOH) in both the potassium superoxide and xanthine oxidase/xanthine systems, indicating that resveratrol could directly scavenge superoxide anions. The inhibition of DEPMPO-OOH in the xanthine oxidase/xanthine system, however, was found to be much potent as compared to that observed in potassium superoxide system. It was further shown that resveratrol could also directly inhibit xanthine oxidase activity as assessed by oxygen consumption and formation of uric acid. Taken together, the dual role of resveratrol in directly scavenging superoxide and inhibiting its generation via xanthine oxidase reported in this study may explain, at least in part, the protective role of this compound against oxidative injury in various disease processes.     

A sensitive fluorometric assay for measuring xanthine dehydrogenase and oxidase in tissues

The conversion of xanthine dehydrogenase to a free radical producing oxidase is an important component of oxygen-mediated tissue injury. Current assays for these enzymes are of limited sensitivity, making it difficult to analyze activities in organ biopsies or cultured cells. The xanthine oxidase-catalyzed conversion of pterin (2-amino-4-hydroxypteridine) to isoxanthopterin provides the basis for a fluorometric assay which is 100-500 times more sensitive than the traditional spectrophotometric assay of urate formation from xanthine. Enzyme activity as low as 0.1 pmol min-1 ml-1 can be measured with the fluorometric pterin assay. Xanthine oxidase is assayed in the presence of pterin only, while combined xanthine dehydrogenase plus oxidase activity is determined with methylene blue which replaces NAD+ as an electron acceptor. The relative proportions and specific activities of xanthine oxidase and dehydrogenase determined by the fluorometric pterin assay are comparable with the spectrophotometric measurement of activities present in rat liver, intestine, kidney, and plasma. The assay has been successfully applied to brain, human kidney, and cultured mammalian cells, where xanthine dehydrogenase and oxidase activities are too low to detect spectrophotometrically.     

Further evidence for the role of free radicals in the limb teratogenicity of L-NAME

BACKGROUND: L-NAME (N(G)-nitro-(L)-arginine methyl ester), a nitric oxide synthase inhibitor, causes severe limb reduction malformations when gravid rats are treated intraperitoneally on gd-17. Hemorrhages, appearing within hours of L-NAME administration, and defects at term can be significantly reduced by co-treatment with PBN (alpha-phenyl-N-t-butylnitrone), a spin trap antioxidant. We have proposed that limb defects result from ischemia-reperfusion injury. We examine the role of xanthine oxidase and ROS formation in the limb effects of L-NAME. METHODS: Gravidas were treated with L-NAME (50 mg/kg) in the presence or absence of allopurinol, a xanthine oxidase inhibitor. Spatial patterns of limb hemorrhage were determined promptly and at term as was digit length at the latter interval. Xanthine oxidase activities were assayed in control and treated limbs with and without allopurinol co-treatment. RESULTS: Allopurinol significantly reduced hemorrhage severity in a dose-responsive fashion when fetuses were examined at term. Higher doses of allopurinol significantly preserved digit length. Xanthine oxidase activities in fetal limb were significantly increased by L-NAME treatment whereas co-treatment with allopurinol restored activities to near-control levels. CONCLUSIONS: These findings support the role of excess reactive oxygen species (ROS) formation in L-NAME-induced limb reduction. We propose that nitric oxide (NO) depletion by L-NAME interferes with vascular integrity, and causes vasoconstriction. Resultant hypoxia stimulates superoxide formation and nitric oxide formation catalyzed by the inducible isoform of nitric oxide synthase. The reduction products of superoxide or the products of its reaction with nitric oxide oxidize or nitrate endothelial components resulting in limb reduction defects.     

Xanthine oxidase/hydrogen peroxide generates sulfur trioxide anion radical (SO3.-) from sulfite (SO3(2-)).

In the presence of hydrogen peroxide (H2O2), xanthine oxidase has been found to catalyze sulfur trioxide anion radical (SO3.-) formation from sulfite anion (SO3(2-)). The SO3.- radical was identified by ESR (electron spin resonance) spin trapping, utilizing 5,5-dimethyl-l-pyrroline-l-oxide (DMPO) as the spin trap. Inactivated xanthine oxidase does not catalyze SO3.- radical formation, implying a specific role for this enzyme. The initial rate of SO3.- radical formation increases linearly with xanthine oxidase concentration. Together, these observations indicate that the SO3.- generation occurs enzymatically. These results suggest a new property of xanthine oxidase and perhaps also a significant step in the mechanism of sulfite toxicity in cellular systems.     

Synthesis of resveratrol analogues, and evaluation of their cytotoxic and xanthine oxidase inhibitory activities

Thirteen resveratrol (=5-[(E)-2-(4-hydroxyphenyl)ethenyl]benzene-1,3-diol) analogues with a CHO group have been prepared by partial synthesis from resveratrol. The synthesized compounds have been evaluated for their cytotoxic activity against a human nasopharyngeal epidermoid tumor cell line KB, as well as for their xanthine oxidase inhibitory activity. Compounds 2, 3, and 6a showed the most significant cytotoxic activities against the cell line KB, and compound 2 also exhibited strong xanthine oxidase inhibitory activity.     

Bioflavonoids as antiradicals,antioxidants and DNA cleavage protectors

Flavonoids have recently aroused considerable interest because of their broad pharmacological activity. In fact, flavonoids have been reported to have antiviral, antiallergic, antiplatelet, anti-inflammatory and antitumoral activities. The pharmacological properties of bioflavonoids have been ascribed both to the concomitant inhibition of enzymes involved in the production of free radicals and to their free-radical scavenging and iron chelating capacity. However the antioxidant capacity of bioflavonoids due to free-radical scavenging and/or to iron chelating is still controversial. In this study, we have investigated the free-radical scavenging capacity of bioflavonoids (rutin, catechin, and naringin). In addition, the effects of these polyphenols on xanthine oxidase activity, spontaneous lipid peroxidation, and DNA cleavage were investigated. The bioflavonoids under examination showed a dose-dependent free-radical scavenging effect, a significant inhibition of xanthine oxidase activity, and an antilipoperoxidative capacity. In addition, they showed a protective effect on DNA cleavage. This revised version was published online in July 2006 with corrections to the Cover Date.     

Biochemical characterization of MelJ and MelK

Melithiazol and myxothiazol are two myxobacterial metabolites that are highly efficient electron transport inhibitors of the respiratory chain. MelJ and MelK encoded in the melithiazol biosynthetic gene cluster were recently shown to be involved in the formation of the methyl ester from a hypothetical amide intermediate. In vivo studies suggest that the structurally highly similar amide myxothiazol A can be used as a substrate mimic of the hypothetical melithiazol amide to characterize the hydrolase MelJ. Both enzymes were produced in Escherichia coli as intein chitin fusion proteins and were purified using affinity chromatography. MelJ was found to catalyse the conversion of the amide myxothiazol to free myxothiazol acid. The formerly unknown myxothiazol acid was purified and used as a substrate for the methyl transferase MelK which methylates the compound using S-adenosyl-methionine as cosubstrate. Sequence analyses suggest that MelJ and MelK are members of the amidase signature family and of a new subclass of methyltransferases, respectively. Kinetic analyses point at a very high substrate specificity for both enzymes. Furthermore, the in vitro reconstitution of a unique mechanism of methyl ester formation found in myxobacteria is reported.     

Molybdenum and tungsten enzymes: the xanthine oxidase family

Mononuclear molybdenum and tungsten are found in the active site of a diverse group of enzymes that, in general, catalyze oxygen atom transfer reactions. Enzymes of the xanthine oxidase family are the best-characterized mononuclear Mo-containing enzymes. Several 3D structures of diverse members of this family are known. Recently, the structures of substrate-bound and arsenite-inhibited forms of two members of this family have also been reported. In addition, spectroscopic studies have been utilized to elucidate fine details that complement the structural information. Altogether, these studies have provided an important amount of information on the characteristics of the active site and the electron transfer pathways.     

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.     

Peroxynitrite formation from the simultaneous reduction of nitrite and oxygen by xanthine oxidase

One electron reductions of oxygen and nitrite by xanthine oxidase form peroxynitrite. The nitrite and oxygen reducing activities of xanthine oxidase are regulated by oxygen with K(oxygen) 26 and 100 microM and K(nitrite) 1.0 and 1.1 mM with xanthine and NADH as donor substrates. Optimal peroxynitrite formation occurs at 70 microM oxygen with purine substrates. Kinetic parameters: V(max) approximately 50 nmol/min/mg and K(m) of 22, 36 and 70 microM for hypoxanthine, pterin and nitrite respectively. Peroxynitrite generation is inhibited by allopurinol, superoxide dismutase and diphenylene iodonium. A role for this enzyme activity can be found in the antibacterial activity of milk and circulating xanthine oxidase activity.     

Reactive oxygen species (ROS)-generating oxidases in the normal rabbit cornea and their involvement in the corneal damage evoked by UVB rays

The corneas of albino rabbits were irradiated (5 min exposure once a day) with UVB rays (312 nm) for 4 days (shorter procedure) or 8 days (longer procedure). The eyes were examined microbiologically and only the corneas of sterile eyes or eyes with non-pathogenic microbes were employed. Histochemically, the activities of reactive oxygen species (ROS)-generating oxidases (xanthine oxidase, D-amino acid oxidase and alpha-hydroxy acid oxidase) were examined in cryostat sections of the whole corneas. Biochemically, the activity of xanthine oxidoreductase/xanthine oxidase was investigated in the scraped corneal epithelium. UVB rays significantly changed enzyme activities in the corneas. In comparison to the normal cornea, where of ROS-generating oxidases only xanthine oxidase showed significant activity in the corneal epithelium and endothelium, D-amino acid oxidase was very low and alpha-hydroxy acid oxidase could not be detected at all, in the cornea repeatedly irradiated with UVB rays, increased activities of xanthine oxidase and D-amino acid oxidase were observed in all corneal layers. Only after the longer procedure the xanthine oxidase and D-amino acid oxidase activities were decreased in the thinned epithelium in parallel with its morphological disturbances. Further results show that the xanthine oxidase/xanthine oxidoreductase ratio increased in the epithelium together with the repeated irradiation with UVB rays. This might suggest that xanthine dehydrogenase is converted to xanthine oxidase. However, in comparison to the normal corneal epithelium, the total amount of xanthine oxidoredutase was decreased in the irradiated epithelium. It is presumed that xanthine oxidoreductase might be released extracellularly (into tears) or the enzyme molecules were denatured due to UVB rays (particulary after the longer procedure). Comparative histochemical and biochemical findings suggest that reactive oxygen species-generating oxidases (xanthine oxidase, D-amino acid oxidase) contribute to the corneal damage evoked by UVB rays.     

Relationship between mechanical dysfunction and depression of sarcolemmal Ca2+-pump activity in hearts perfused with oxygen free radicals

Although in vitro studies have shown that oxygen free radicals depress the sarcolemmal Ca²⁺-pump activity and thereby may cause the occurrence of intracellular Ca²⁺ overload for the genesis of contractile failure, the exact relationship between changes in sarcolemmal Ca²⁺-pump activity and cardiac function due to these radicals is not clear. In this study we examined the effects of oxygen radicals on sarcolemmal Ca²⁺ uptake and Ca²⁺-stimulated ATPase activities as well as contractile force development by employing isolated rat heart preparations. When hearts were perfused with medium containing xanthine plus xanthine oxidase, the sarcolemmal Ca²⁺-stimulated ATPase activity and ATP-dependent Ca²⁺ accumulation were depressed within 1 min whereas the developed contractile force, rate of contraction and rate of relaxation were increased at 1 min and decreased over 3–20 min of perfusion. The resting tension started increasing at 2 min of perfusion with xanthine plus xanthine oxidase. Catalase showed protective effects against these alterations in heart function and sarcolemmal Ca²⁺-pump activities upon perfusion with xanthine plus xanthine oxidase whereas superoxide dismutase did not exert such effects. The combination of catalase and superoxide dismutase did not produce greater effects in comparison to catalase alone. These results are consistent with the view that the depression of heart sarcolemmal Ca²⁺ pump activities may result in myocardial dysfunction due to the formation of hydrogen peroxide and/or hydroxyl radicals upon perfusing the hearts with xanthine plus xanthine oxidase.     

Mo and W bis-MGD enzymes: nitrate reductases and formate dehydrogenases

Molybdenum and tungsten are second- and third-row transition elements, respectively, which are found in a mononuclear form in the active site of a diverse group of enzymes that generally catalyze oxygen atom transfer reactions. Mononuclear Mo-containing enzymes have been classified into three families: xanthine oxidase, DMSO reductase, and sulfite oxidase. The proteins of the DMSO reductase family present the widest diversity of properties among its members and our knowledge about this family was greatly broadened by the study of the enzymes nitrate reductase and formate dehydrogenase, obtained from different sources. We discuss in this review the information of the better characterized examples of these two types of Mo enzymes and W enzymes closely related to the members of the DMSO reductase family. We briefly summarize, also, the few cases reported so far for enzymes that can function either with Mo or W at their active site.     

2-styrylchromones as novel inhibitors of xanthine oxidase. A structure-activity study

The purpose of this study was the evaluation of the xanthine oxidase (XO) inhibition produced by some synthetic 2-styrylchromones. Ten polyhydroxylated derivatives with several substitution patterns were synthesised, and these and a positive control, allopurinol, were tested for their effects on XO activity by measuring the formation of uric acid from xanthine. The synthesised 2-styrylchromones inhibited xanthine oxidase in a concentration-dependent and non-competitive manner. Some IC50 values found were as low as 0.55 microM, which, by comparison with the IC50 found for allopurinol (5.43 microM), indicates promising new inhibitors. Those 2-styrylchromones found to be potent XO inhibitors should be further evaluated as potential agents for the treatment of pathologies related to the enzyme's activity, as is the case of gout, ischaemia/reperfusion damage, hypertension, hepatitis and cancer.     

2-Styrylchromones As Novel Inhibitors of Xanthine Oxidase. A Structure-activity Study

The purpose of this study was the evaluation of the xanthine oxidase (XO) inhibition produced by some synthetic 2-styrylchromones. Ten polyhydroxylated derivatives with several substitution patterns were synthesised, and these and a positive control, allopurinol, were tested for their effects on XO activity by measuring the formation of uric acid from xanthine. The synthesised 2-styrylchromones inhibited xanthine oxidase in a concentration-dependent and non-competitive manner. Some IC 50 values found were as low as 0.55 μM, which, by comparison with the IC 50 found for allopurinol (5.43 μM), indicates promising new inhibitors. Those 2-styrylchromones found to be potent XO inhibitors should be further evaluated as potential agents for the treatment of pathologies related to the enzyme's activity, as is the case of gout, ischaemia/reperfusion damage, hypertension, hepatitis and cancer.     

Effect of exogenous and endogenous nitrosation factors on formation of N-nitrosodimethylamine in rats depending on the status of purine catabolism

Nitrogen dioxide's rats' inhalations with injections per os of pyrazole, amidopyrine and sodium nitrite lead to considerable increasing of endogenic N-nitrosodimethylamine formation, which had been determined by system gas chromatograph-thermal energetic analyser. This increasing essentially didn't depend on the rats' immunisation by vaccine BCG, which leads to the intensification of NO synthesis by peritoneal macrophages and others manifestations of their metabolic activation: increasing of creatine kinase and adenosine desaminase activities. It hadn't been brought to light the obvious dependent between changes of xanthine oxidase and xanthine dehydrogenase activities in the liver and blood serum and intensification of lipids peroxidation and also the amount of N-nitrosodimethylamine in the rats in the conditions of endogenic and exogenic nitrosation factors' influence.     

Application of polyethylene glycol-modified enzymes in biotechnological processes: organic solvent-soluble enzymes

A new approach in biotechnological processes is to use enzymes modified with polyethylene glycol which has both hydrophilic and hydrophobic properties. The modified enzymes are soluble in organic solvents such as benzene, toluene and chlorinated hydrocarbons and exhibit high enzymic activities in these organic solvents. Modified hydrolytic enzymes catalysed the reverse reaction of hydrolysis in organic solvents: formation of acid—amide bonds by modified chymotrypsin, and ester synthesis and ester exchange reactions by modified lipase. Modified catalase and modified peroxidase efficiently catalyse their respective reactions in organic solvents. The results of this research indicate great potential for applications in the fields of biotechnology and enzymology.     

Novel 3-O-acyl mesquitol analogues as free-radical scavengers and enzyme inhibitors: synthesis,biological evaluation and structure-activity relationship

A new isomer of mesquitol (2,3-trans-3',4',7,8-tetrahydroxyflavan-3-ol) was isolated from Dichrostachys cinerea in excellent yields. It has shown free-radical scavenging property and alpha-glucosidase inhibitory activities but, it could not display xanthine oxidase inhibitory property. However, it was observed that acylation of 3-OH group significantly enhanced the alpha-glucosidase inhibition and displayed xanthine oxidase inhibitory potential. The structure activity relationship revealed that the degree of lipophilicity played a major role in improving enzyme inhibitory activities. A positive correlation was observed between enzyme inhibitory potential and acyl chain length (upto C-16) of aliphatic esters.