Xanthine:NAD+ oxidoreductase in the liver of grass snake Natrix natrix

The enzyme hydroxylating oxypurines in the liver of grass snake (Natrix natrix, Colubridae) was found to be a stable xanthine:NAD+ oxidoreductase (EC 1.2.1.37). The Michaelis constants for NAD+ and xanthine amounted to 14.4 and 12.3 microM, respectively. The enzyme affinity to hypoxanthine is lower than that to xanthine, but the former substrate is hydroxylated faster than the latter. The enzyme is only slowly and slightly (up to 22%) inhibited by NADH accumulating during xanthine hydroxylation. The above data and the time-course of hypoxanthine----xanthine----uric acid hydroxylation indicated that the kinetic properties of the snake liver enzyme provide in this uricotelic animal fast elimination of superfluous nitrogen derived from protein catabolism.     

Xanthine:NAD+ oxidoreductase from embryo liver of hen Gallus gallus

1. Xanthine:NAD+ oxidoreductase from chick embryo liver is unconvertible to the O2-dependent form, as is the enzyme from the adult hen. The Km for NAD+ (approximately 3 microM) of the embryonic enzyme is equal to, and the Km for xanthine (approximately 5 microM) is 2.5-fold lower, when compared with respective Km values of the "adult" hen enzyme. The inhibition of embryonic enzyme by NADH begins at 10 microM NADH and attains 13% at 35 microM NADH (respective data for the "adult" enzyme: 50 microM and 20% at 80 microM NADH). 2. The course of hypoxanthine----xanthine----uric acid hydroxylation catalyzed by the embryonic and "adult" enzymes is similar, however the rate of the first reaction is 2-fold lower for the embryonic enzyme. Under conditions of the limited nutritional system in the developing chick embryo, the low rate of hypoxanthine hydroxylation may promote reutilization of hypoxanthine for nucleotide synthesis.     

Effect of NADH on hypoxanthine hydroxylation by native NAD+-dependent xanthine oxidoreductase of rat liver, and the possible biological role of this effect.

The course of the reaction sequence hypoxanthine leads to xanthine leads to uric acid, catalysed by the NAD+-dependent activity of xanthine oxidoreductase, was investigated under conditions either of immediate oxidation of the NADH formed or of NADH accumulation. The enzymic preparation was obtained from rat liver, and purified 75-fold (as compared with the 25000 g supernatant) on a 5'-AMP-Sepharose 4B column; in this preparation the NAD+-dependent activity accounted for 100% of total xanthine oxidoreductase activity. A spectrophotometric method was developed for continuous measurements of changes in the concentrations of the three purines involved. The time course as well as the effects of the concentrations of enzyme and of hypoxanthine were examined. NADH produced by the enzyme lowered its activity by 50%, resulting in xanthine accumulation and in decreases of uric acid formation and of hypoxanthine utilization. The inhibition of the Xanthine oxidoreductase NAD+-dependent activity by NADH is discussed as a possible factor in the regulation of IMP biosynthesis by the 'de novo' pathway or (from unchanged hypoxanthine) by ther salvage pathway.     

Purification and properties of xanthine dehydrogenase from Streptomyces cyanogenus.

Xanthine dehydrogenase has been purified to a homogeneous state from cell-free extracts of a strain of Streptomyces. The enzyme has a molecular weight of 125,000 and consists of two subunits with a molecular weight of 67,000. The isoelectric point is at pH 4.4. The enzyme exhibits absorption maxima at 273, 355, and 457 nm and contains FAD, iron, and labile sulfide in a molar ratio of 1 : 7 : 1 per subunit. Little molybdenum could be detected. The enzyme is most active at pH 8.7 and at 40 degrees C, and is stable between pH 7 and 12 (at 4 degrees C for 24 h) and below 55 degrees C (at pH 9 for 10 min). The activity is stimulated by K+ at a concentration of 50 mM or more and also by keeping the enzyme at pH 9 to 11. The activity is inhibited by cyanide, Tiron, and p-chloromercuribenzoate and by adenine and urate. Among the compounds tested, hypoxanthine, guanine, xanthine 2-hydroxypurine, and 6,8-dihydroxypurine are oxidized at considerable rates; hypoxanthine is the best substrate. NAD+ is the preferred electron acceptor. Km values of the enzyme for hypoxanthine, guanine, xanthine, and NAD+ are 0.055, 0.015, 0.15, and 0.11 mM, respectively. Marked differences in the properties of this enzyme compared to others are the activity towards guanine, which has a higher affinity for the enzyme than hypoxanthine and xanthine, and a higher reactivity with hypoxanthine than xanthine. The organism has been identified as Streptomyces cyanogenus.     

Comparison of xanthine: NAD+ oxidoreductase from liver of toad Bufo viridis and other vertebrates

1. Xanthine oxidoreductase was isolated from toad Bufo viridis (a mainly ureotelic amphibian species) and partially purified. The enzyme occurred as a stable xanthine: NAD+ oxidoreductase (EC 1.1.1.204), unconvertible to the oxidase form. 2. Some properties of the enzyme resembled those of xanthine oxidoreductase from an ammonotelic fish, Cyprinus carpio, and the ureotelic rat, but in other aspects it was similar to this enzyme from an uricotelic snake, Natrix natrix. 3. Inhibition of the toad enzyme by NADH at high non-physiological concentrations rules out a modulation of its oxypurine-hydroxylating activity by in vivo changes in the NADH/NAD+ ratio. Therefore, toad xanthine oxidoreductase plays no regulatory role in the purine nucleotide metabolism.     

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.     

Soybean nodule xanthine dehydrogenase: a kinetic study

Xanthine dehydrogenase was purified from soybean nodules and the kinetic properties were studied at pH 7.5. Km values of 5.0 +/- 0.6 and 12.5 +/- 2.5 microM were obtained for xanthine and NAD+, respectively. The pattern of substrate dependence suggested a Ping-Pong mechanism. Reaction with hypoxanthine gave Km's of 52 +/- 3 and 20 +/- 2.5 microM for hypoxanthine and NAD+, respectively. The Vmax for this reaction was twice that for the xanthine-dependent reaction. The pH dependence of Vmax gave a pKa of 7.6 +/- 0.1 for either xanthine or hypoxanthine oxidation. In addition the Km for xanthine had a pKa of 7.5 consistent with the protonated form of xanthine being the true substrate. Km for hypoxanthine varied only 2.5-fold between pH 6 and 10.7. Product inhibition studies were carried out with urate and NADH. Both products gave mixed inhibition with respect to both substrates. Xanthine dehydrogenase was able to use APAD+ as an electron acceptor for xanthine oxidation, with a Km at pH 7.5 of 21.2 +/- 2.5 microM and Vmax the same as that obtained with NAD+. Reduction of APAD+ by NADH was also catalyzed by xanthine dehydrogenase with a Km of 102 +/- 15 microM; Vmax was approximately 2.5 times that for the xanthine-dependent reaction, and was independent of pH between 6 and 9. Reaction with group-specific reagents indicated the possibility of an essential histidyl group. A thiol-modifying reagent did not cause inactivation of the enzyme. A role for the histidyl side chain in catalysis is proposed.     

Purification and properties of xanthine dehydrogenase from Pseudomonas acidovorans.

Xanthine dehydrogenase (EC 1.2.1.37) from Pseudomonas acidovorans has been purified to near homogeneity (approx. 65-fold). The enzyme has a molecular weight of about 275 000. Electrophoresis in gels containing sodium dodecyl sulphate showed the presence of two types of subunit with molecular weights of about 81 000 and 63 000. Thus the intact molecule probably contains two of each type of subunit. Xanthine and hypoxanthine are good substrates, and NAD+ is an effective electron acceptor. With xanthine and NAD+ as substrates the purified enzyme has a specific activity of about 20 mumol NADH formed/min per mg protein. Michaelis constants for xanthine and NAD+ are 0.07 and 0.12 mM, respectively, and for hypoxanthine and NAD+ 0.29 and 0.16 mM, respectively.     

Purification and properties of purine hydroxylase II from Aspergillus nidulans

Purine hydroxylase II from Aspergillus nidulans has been purified to near homogeneity. The enzyme has a pI of 5.7, a molecular weight of 300,000, and two subunits with molecular weight of 153,000 each. The enzyme contains 2 FAD, 2 molybdenum atoms, and 4 (2 Fe-2S) iron-sulfur centers per molecule and exhibits broad specificity for reducing and oxidizing substrates. Among the more notable characteristics are the ability to oxidize hypoxanthine and nicotinic acid but not xanthine and virtually complete inactivity with oxygen. Moreover, while the enzyme is inactivated by borate and methanol, it is very resistant to cyanide and arsenite and it not inactivated by allopurinol. At infinite concentrations of reducing and oxidizing substrates, the Km for hypoxanthine was 119 microM, for nicotinic acid was 136 microM, and for NAD+ was 525 microM.     

Crystal structures of the active and alloxanthine-inhibited forms of xanthine dehydrogenase from Rhodobacter capsulatus.

Xanthine dehydrogenase (XDH), a complex molybdo/iron-sulfur/flavoprotein, catalyzes the oxidation of hypoxanthine to xanthine followed by oxidation of xanthine to uric acid with concomitant reduction of NAD+. The 2.7 A resolution structure of Rhodobacter capsulatus XDH reveals that the bacterial and bovine XDH have highly similar folds despite differences in subunit composition. The NAD+ binding pocket of the bacterial XDH resembles that of the dehydrogenase form of the bovine enzyme rather than that of the oxidase form, which reduces O(2) instead of NAD+. The drug allopurinol is used to treat XDH-catalyzed uric acid build-up occurring in gout or during cancer chemotherapy. As a hypoxanthine analog, it is oxidized to alloxanthine, which cannot be further oxidized but acts as a tight binding inhibitor of XDH. The 3.0 A resolution structure of the XDH-alloxanthine complex shows direct coordination of alloxanthine to the molybdenum via a nitrogen atom. These results provide a starting point for the rational design of new XDH inhibitors.     

Oxidative stress to human lymphocytes by xanthine oxidoreductase activity

The in vitro toxicity of the reactive oxygen species generating enzyme xanthine oxidoreductase (XOR) to human peripheral blood lymphocytes was studied after stimulation with phytohaemoagglutinin or anti-CD3/CD28 antibodies. Apoptosis and necrosis were induced by the XOR/hypoxanthine system in a time- and concentration-dependent manner. CD8+ lymphocytes showed a higher sensitivity than CD4+ cells to the XOR/hypoxanthine system. The occurrence of apoptosis was demonstrated by annexin-V binding to injured cell membrane, which was the most precocious alteration observed, followed by the increment of transglutaminase activity, which was significant at the lowest XOR concentration used. Nuclear damage was assessed by the increased hypodiploid nuclei and by DNA migration on gel electrophoresis, which turned to an apoptotic pattern before the occurrence of cell membrane necrotic lesions. Apoptosis was induced by XOR activity proportionally to substrate concentration and was prevented by the competitive enzyme inhibitor, allopurinol. The hydrogen peroxide scavenging enzyme, catalase, gave a higher protection than superoxide dismutase from the toxicity caused by the XOR/hypoxanthine system. Necrosis occurs in a variable percentage indicating that reactive oxygen species may trigger both apoptosis and necrosis in proliferating human lymphocytes, mostly depending on XOR concentration.     

Oxidative stress to human lymphocytes by xanthine oxidoreductase activity

The in vitro toxicity of the reactive oxygen species generating enzyme xanthine oxidoreductase (XOR) to human peripheral blood lymphocytes was studied after stimulation with phytohaemoagglutinin or anti-CD3/CD28 antibodies. Apoptosis and necrosis were induced by the XOR/hypoxanthine system in a time- and concentration-dependent manner. CD8+ lymphocytes showed a higher sensitivity than CD4+ cells to the XOR/hypoxanthine system. The occurrence of apoptosis was demonstrated by annexin-V binding to injured cell membrane, which was the most precocious alteration observed, followed by the increment of transglutaminase activity, which was significant at the lowest XOR concentration used. Nuclear damage was assessed by the increased hypodiploid nuclei and by DNA migration on gel electrophoresis, which turned to an apoptotic pattern before the occurrence of cell membrane necrotic lesions. Apoptosis was induced by XOR activity proportionally to substrate concentration and was prevented by the competitive enzyme inhibitor, allopurinol. The hydrogen peroxide scavenging enzyme, catalase, gave a higher protection than superoxide dismutase from the toxicity caused by the XOR/hypoxanthine system. Necrosis occurs in a variable percentage indicating that reactive oxygen species may trigger both apoptosis and necrosis in proliferating human lymphocytes, mostly depending on XOR concentration.     

A new spectrophotometric assay for enzymes of purine metabolism. I. Determination of xanthine oxidase activity.

A new method for the determination of xanthine oxidase activity with xanthine or hypoxanthine is described. The hydrogen peroxide produced by the oxidation of the substrates is reduced by catalase in the presence of high concentrations of ethanol. The acetaldehyde formed is further oxidized by aldehyde dehydrogenase NAD or NADP-dependent. The reduction rate of the coenzymes were measured at 334 nm and utilized as indicators for the xanthine oxidase. The sensitivity of the method with xanthine as substrate can be doubled by the addition of uricase, which oxidizes uric acid to allantoin.     

Activation of nitrite reductase from Escherichia coli K12 by oxidized nicotinamide-adenine dinucleotide.

Nitrite reductase from Escherichia coli K12 requires the presence of NAD+, one of the products of the reduction of NO2-by NADH, for full activity. The effect is observed with both crude extracts and purified enzyme. NAD+ also acts as a product inhibitor at high concentrations, and plots of initial rate against NAD+ concentration are bell-shaped. The maximum occurs at about 1 mM-NAD+, but increases with increasing NADH concentration. In the presence of 1 mM-NAD+ and saturating NO2-(2mM) the Michaelis constant for NADH is about 16 micron. The Michaelis constant for NO2-is about 5 micron and is largely independent of the NAD+ concentration. Similar but more pronounced effects of NAD+ are observed with hydroxylamine as electron acceptor instead of NO2-. The maximum rate of NADH oxidation by hydroxylamine is about 5.4 times greater than the maximum rate of NADH oxidation by NO2- when assayed with the same volume of the same preparation of purified enzyme. The Michaelis constant for hydroxylamine is 5.3 mM, however, about 1000 times higher than for NO2-. These results are consistent with a mechanism in which the same enzyme-hydroxylamine complex occurs as an intermediate in both reactions.     

A kinetic study of hypoxanthine oxidation by milk xanthine oxidase.

The course of the reaction sequence hypoxanthine----xanthine----uric acid catalysed by xanthine:oxygen oxidoreductase from milk was investigated on the basis of u.v. spectra taken during the course of hypoxanthine and xanthine oxidations. It was found that xanthine accumulated in the reaction mixture when hypoxanthine was used as a substrate. The time course of the concentrations of hypoxanthine, xanthine intermediate and uric acid product was simulated numerically. The mathematical model takes into account the competition of substrate, intermediate and product and the accumulation of the intermediate at the enzyme. This type of analysis permits the kinetic parameters of the enzyme for hypoxanthine and xanthine to be obtained.     

Xanthine dehydrogenase from Pseudomonas putida 86: specificity, oxidation-reduction potentials of its redox-active centers, and first EPR characterization

Xanthine dehydrogenase (XDH) from Pseudomonas putida 86, which was induced 65-fold by growth on hypoxanthine, was purified to homogeneity. It catalyzes the oxidation of hypoxanthine, xanthine, purine, and some aromatic aldehydes, using NAD+ as the preferred electron acceptor. In the hypoxanthine:NAD+ assay, the specific activity of purified XDH was 26.7 U (mg protein)(-1). Its activity with ferricyanide and dioxygen was 58% and 4%, respectively, relative to the activity observed with NAD+. XDH from P. putida 86 consists of 91.0 kDa and 46.2 kDa subunits presumably forming an alpha4beta4 structure and contains the same set of redox-active centers as eukaryotic XDHs. After reduction of the enzyme with xanthine, electron paramagnetic resonance (EPR) signals of the neutral FAD semiquinone radical and the Mo(V) rapid signal were observed at 77 K. Resonances from FeSI and FeSII were detected at 15 K. Whereas the observable g factors for FeSII resemble those of other molybdenum hydroxylases, the FeSI center in contrast to most other known FeSI centers has nearly axial symmetry. The EPR features of the redox-active centers of P. putida XDH are very similar to those of eukaryotic XDHs/xanthine oxidases, suggesting that the environment of each center and their functionality are analogous in these enzymes. The midpoint potentials determined for the molybdenum, FeSI and FAD redox couples are close to each other and resemble those of the corresponding centers in eukaryotic XDHs.     

Substrate inhibition of xanthine oxidase and its influence on superoxide radical production.

The influence of substrate inhibition on xanthine oxidase-intramolecular electron transport was studied by steady-state kinetic analysis. Experiments with hypoxanthine and xanthine up to 900 microM indicated an inhibition pattern which fitted an equation of the general form nu 0 = nu max . [S]/(Km + a[S] + b[S]2/Ki). Univalent electron flux to oxygen was favored at substrate concentrations above 50 microM. This augmentation of univalent flux percentage that appeared at a high substrate concentration was greater for hypoxanthine that xanthine and at pH 8.3 than at 9.5. Our results support a mechanism of inhibition in which a substrate-reduced enzyme, non-productive Michaelis complex was formed. It is possible that this non-productive complex favored the univalent pathway of enzyme reoxidation (superoxide production) by increasing the midpoint redox potential of the molybdenum active site.     

Involvement of histidine residues in catalytic activity of xanthine dehydrogenase from hen liver.

1. Modification of histidine residue(s) of xanthine dehydrogenase from hen liver by DEP and photooxidation results in loss of the ability to transfer electrons from xanthine to NAD+ and also from NADH to 2,6-dichlorophenolindophenol (DCIP). 2. The kinetics of inactivation suggest that carbethoxylation of more than one histidyl residue in the enzyme may be responsible for the inactivation.     

Effects of long-term beer ingestion on plasma concentrations and urinary excretion of purine bases.

To investigate the long-term effects of beer ingestion on plasma concentrations of purine bases (hypoxanthine, xanthine, and uric acid), ten healthy males ingested beer (15 ml/kg body weight) every evening for three months. Blood and 24-hour urine samples were collected in the morning on one day before and one, two, and three months after starting the experiment to determine the plasma concentrations and urinary excretion of uric acid, hypoxanthine, and xanthine. Plasma concentrations and urinary excretion of uric acid, hypoxanthine, and xanthine in five of the participants that did not regularly ingest beer at a quantity of more than 15 ml/kg body weight in a single day prior to the experiment were not increased during the experimental period. In contrast, plasma concentrations and urinary excretion of uric acid were increased in five participants who regularly ingested more than 15 ml/kg body weight of beer in a single day prior to the experiment, although hypoxanthine and xanthine levels were not significantly increased during the experimental period. In both groups, uric acid clearance and purine ingestion were not significantly different throughout the study. Our results suggest that the production of uric acid caused by ethanol ingestion from beer is a significant contributor to the increase in plasma uric acid concentration in patients that regularly consume more than 15 ml/kg body weight of beer each day. Therefore, patients with gout should be encouraged to refrain from drinking large amounts of beer on a daily basis.     

Involvement of a single thiol group in the conversion of the NAD+-dependent activity of rat liver xanthine oxidoreductase to the O2-dependent activity.

The effects of 2-iodosobenzoic acid, 4-chloromercuribenzoate, 5,5'-dithiobis-(2-nitrobenzoic acid) and tetraethylthioperoxydicarbonic diamide (disulphiram) on the NAD+-dependent activity of xanthine oxidoreductase from rat liver were investigated. Only disulphiram converted the NAD+-dependent activity into the O2-dependent activity quantitatively, without changing the xanthine hydroxylation rate. The modification process was a first-order reaction with respect to time (min) and disulphiram concentration (microM). The kinetic data showed that modification of single thiol group is sufficient for loss of the enzymic activity towards NAD+ as electron acceptor. The complete protection afforded by NAD+ against the action of disulphiram suggests that the essential thiol group may be involved in binding of NAD+ to the xanthine oxidoreductase molecule.