Purification and characterization of spiralin, the main protein of the Spiroplasma citri membrane

1. Hypoxanthines, bearing at position 8 aryl or pyridyl substituents, are converted by bovine milk xanthine oxidase (xanthine: oxygen oxidoreductase, EC 1.2.3.2) into the corresponding xanthines at low rates. Oxidation is accelerated considerably when the 8-pyridyl substituents are quaternised. 2. In the enzymic oxidation of quaternary 8-pyridylhypoxanthines a lag phase precedes the attainment of a constant, maximal reaction rate. It is assumed that the delay is due to a relatively slow conformational change in the active enzymic center. 3. In 8-(3'-N-methylpyridinio)xanthine betaine, also the pyridinium moiety is attacked at high pH (9-11) to yield an N-methyl-2-pyridone. The analogous pyridone is the only oxidation product of 1-methyl-8-(3'-N-methylpyridinio)-hypoxanthine betaine, which is not attacked in the pyrimidine ring. 4. The cationic substrates are attracted to the enzyme by an anionic group, which probably forms an ion pair with a protonated amino group in or near the active center.     

Oxidation of N-methyl substituted hypoxanthines, xanthines, purine-6,8-diones and the corresponding 6-thioxo derivatives by bovine milk xanthine oxidase.

1. The oxidation of six series of purines (hypoxanthines, xanthines, purine-6,8-diones and the corresponding 6-thioxo derivatives) by a highly purified bovine milk xanthine oxidase (EC 1.2.3.2) has been studied, using a variety of N-methyl derivatives. 2. N-Methyl substituents can either enhance or reduce enzymic rates. Enhancement is ascribed to blockade of groups which mediate unfavorable modes of binding of substrate to enzyme. Introduction of N-methyl groups can also inhibit enzymic oxidation, either by occluding essential binding groups or by preventing spontaneous or enzyme-induced tautomerisation processes, which create suitable binding sites in the substrates. 3. In all purines which are rapidly attacked by xanthine oxidase, proper attachment to the active center is mediated by the groupings (3) NH, (9) N or (3) N, (9) NH. 4. Reduced rates usually express lowered substrate affinity, which finds its expression in weak competitive inhibition of xanthine oxidation.     

Behavior of N-methylated allopurinols and related 4-thioxopyrazolo [3,4-d]pyrimidines towards bovine milk xanthine oxidase.

1. All available N-mono- and N,N'-dimethylallopurinols and the corresponding 4-thioxo derivatives have been tested as substrates or inhibitors of bovine milk xanthine oxidase (xanthine: oxygen oxidoreductase, EC 1.2.3.2). 2. None of the compounds tested revealed any inhibitory activity towards the enzyme. 3. All compounds were resistant to enzymic oxidation, with the exception of 7-methylallopurinol and its 4-thioxo analog. Both these compounds were attacked at position 6. 7-Methylallopurinol was oxidised nearly ten times faster than the isomeric 3-methylhypoxanthine. 4. These observations can be explained by assuming that for attack at C-6, the enzyme must bind both to N-1 and N-2 in the pyrazole ring and causes tautomerisation, which places a double bond at position 5,6 in the pyrimidine ring. This activation process resembles the activation of hypoxanthine.     

Synthesis and preliminary evaluation of new 1- and 3-[1-(2-hydroxy-3-phenoxypropyl)]xanthines from 2-amino-2-oxazolines as potential A1 and A2A adenosine receptor antagonists

The development of potent and selective adenosine receptor ligands as potential drugs is an active area of research. Xanthines are one of the most important classes of adenosine receptor antagonists and have been widely developed in terms of affinity and selectivity for adenosine receptors. We recently developed new original pathways for the synthesis of xanthine analogues starting from 5-substituted-2-amino-2-oxazoline 5 as a synthon. These procedures allowed us to selectively introduce a large, functionalized and beta-adrenergic 2-hydroxy-3-phenoxypropyl pharmacophore at the 1- and 3-position of the xanthine moiety which allowed further structural modifications. In this study, we present a new synthetic access to racemic xanthine derivatives 1-4 from 5, and their evaluation as adenosine A1, A2A and A3 receptor ligands in radioligand binding studies. The 2-hydroxy-3-phenoxypropyl moiety was well tolerated in the 3-position of the xanthine core, while its introduction in the 1-position of the xanthine moiety led to a large decrease in adenosine receptor affinity. 1,7-Dimethyl-3-[1-(2-chloro-3-phenoxypropyl)]-8-(3,4,5-trimethoxystyryl)xanthine (2n) was the most potent and selective A2A antagonist of the present series (Ki=44 nM, >200-fold selective vs A1). 1-Propyl-3-[1-(2-hydroxy-3-phenoxypropyl)]-8-noradamantylxanthine (3f) was identified as a potent (KiA1=21 nM) and highly selective (>350-fold vs A2A and A3 receptor) adenosine A1 receptor antagonist.     

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

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

A molybdopterin-free form of xanthine oxidase

A previously unidentified fraction lacking xanthine:O2 activity has been isolated during affinity chromatography of bovine milk xanthine oxidase preparations on Sepharose 4B/folate gel. Unlike active, desulfo, or demolybdo forms of xanthine oxidase, this form, which typically comprises about 5% of an unfractionated enzyme solution, passes through the affinity column without binding to it, and is thus easily separated from the other species. The absorption spectrum of this fraction is very similar to that of the active form, but has a 7% lower extinction at 450 nm. Analysis of the fraction has shown that it is a dimer of normal size, but that it does not contain molybdenum or molybdopterin (MPT). The "MPT-free" xanthine oxidase contains 90-96% of the Fe found in active xanthine oxidase, and 100% of the expected sulfide. EPR and absorption difference spectroscopy indicate that the MPT-free fraction is missing approximately half of its Fe/S I centers. The presence of a new EPR signal suggests that an altered Fe/S center may account for the nearly normal Fe and sulfide content. Microwave power saturation parameters for the Fe/S II and Fe/S I centers in the MPT-free fraction are normal, with P1/2 equal to 1000 and 60 mW, respectively. The new EPR signal shows intermediate saturation behavior with a P1/2 = 200 mW. The circular dichroism spectrum of the MPT-free fraction shows distinct differences from that of active enzyme. The NADH:methylene blue activity of the MPT-free fraction is the same as that of active xanthine oxidase which exhibits xanthine:O2 activity, but NADH:cytochrome c and NADH:DCIP activities are diminished by 54 and 37%, respectively.     

Binding of sulfurated molybdenum cofactor to the C-terminal domain of ABA3 from Arabidopsis thaliana provides insight into the mechanism of molybdenum cofactor sulfuration

The molybdenum cofactor sulfurase ABA3 from Arabidopsis thaliana is needed for post-translational activation of aldehyde oxidase and xanthine dehydrogenase by transferring a sulfur atom to the desulfo-molybdenum cofactor of these enzymes. ABA3 is a two-domain protein consisting of an NH(2)-terminal NifS-like cysteine desulfurase domain and a C-terminal domain of yet undescribed function. The NH(2)-terminal domain of ABA3 decomposes l-cysteine to yield elemental sulfur, which subsequently is bound as persulfide to a conserved protein cysteinyl residue within this domain. In vivo, activation of aldehyde oxidase and xanthine dehydrogenase also depends on the function of the C-terminal domain, as can be concluded from the A. thaliana aba3/sir3-3 mutant. sir3-3 plants are strongly reduced in aldehyde oxidase and xanthine dehydrogenase activities due to a substitution of arginine 723 by a lysine within the C-terminal domain of the ABA3 protein. Here we present first evidence for the function of the C-terminal domain and show that molybdenum cofactor is bound to this domain with high affinity. Furthermore, cyanide-treated ABA3 C terminus was shown to release thiocyanate, indicating that the molybdenum cofactor bound to the C-terminal domain is present in the sulfurated form. Co-incubation of partially active aldehyde oxidase and xanthine dehydrogenase with ABA3 C terminus carrying sulfurated molybdenum cofactor resulted in stimulation of aldehyde oxidase and xanthine dehydrogenase activity. The data of this work suggest that the C-terminal domain of ABA3 might act as a scaffold protein where prebound desulfo-molybdenum cofactor is converted into sulfurated cofactor prior to activation of aldehyde oxidase and xanthine dehydrogenase.     

Morin hydrate protects cultured rat glomerular mesangial cells against oxyradical damage

Cultured rat glomerular mesangial cells were damaged when exposed to oxyradicals generated either from xanthine oxidase plus hypoxanthine, or by superoxide radicals formed from menadione. Morin hydrate is an antioxidant extracted from yellow Brazil wood. When morin hydrate was added to cultured rat glomerular mesangial cells which were attacked by oxyradicals generated by xanthine oxidase plus hypoxanthine, the survival time of the cells was doubled. However, this protective effect of morin hydrate was less marked when the cells were attacked by menadione. Note that the protective effects of Trolox which is a polar analogue of vitamin E were miniscule relative to those of morin hydrate with both oxidants.     

Milk xanthine oxidase type D (dehydrogenase) and type O (oxidase). Purification, interconversion and some properties

1. The xanthine oxidase of cow's milk, crude or purified, appears as an oxidase (type O), and can be converted almost completely into a NAD(+)-dependent dehydrogenase (type D) by treatment with dithioerythritol or dihydrolipoic acid, but only to a small extent by other thiols. 2. The D form of the enzyme is inhibited by NADH, which competes with NAD(+). 3. The kinetic constants of the two forms of the enzyme are similar to those of the corresponding forms of rat liver xanthine oxidase. 4. Milk xanthine oxidase is converted into an irreversible O form by pretreatment with chymotrypsin, papain or subtilisin, but only partially with trypsin. 5. The enzyme as purified shows a major faster band and a minor slower band on gel electrophoresis. The slower band is greatly reinforced after xanthine oxidase is converted into the irreversible O form by chymotrypsin.     

Absorption and metabolism of purines by the lower intestine of the chicken.

1. Absorption of purines and their metabolism by the lower intestine were estimated by using the everted gut sacs from the colo-rectum and caecum of the chicken. 2. Adenine, hypoxanthine and uric acid were appreciably absorbed from the colo-rectum and caecum, and an especially high rate was observed in the absorption of uric acid from the colo-rectum. 3. Guanine was not absorbed unchanged from either the colo-rectum or the caecum and a small amount of xanthine was absorbed only from the caecum. 4. Hypoxanthine was also absorbed in uric acid form, to a much lesser extent, in xanthine form from the colo-rectum and caecum, adenine and xanthine in uric acid form from the colo-rectum and adenine in hypoxanthine form from the colo-rectum and caecum. 5. Adenine was metabolized to hypoxanthine and xanthine, guanine and hypoxanthine to uric acid and xanthine, and xanthine to adenine, in both mucosal fluids of the colo-rectum and caecum. The conversion of guanine to uric acid in the caecum was most active, being almost twice as much as that in the colo-rectum.     

Role of iron and ethylenediaminetetraacetic acid in the bactericidal activity of a superoxide anion-generating system

In an earlier study, an enzymic superoxide anion-generating system consisting of acetaldehyde plus xanthine oxidase was found to be toxic to Staphylococcus aureus. Both superoxide anion (O

) and its dismutation product hydrogen peroxide (H₂O₂) were required and it was proposed that (O

) and H₂O₂ interact to form the more powerful bactericidal agent(s), hydroxyl radical (OH·) and/or singlet oxygen. Iron chelated by EDTA appears to be a heretofore unrecognized requirement for the xanthine oxidase bactericidal system. The evidence is as follows: (1) the addition of iron salts to the xanthine oxidase system increased bactericidal activity whereas the iron chelators diethylenetriaminepentaacetic acid (DTPA) and desferrioxamine were inhibitory; (2) dialysis of the EDTA-containing xanthine oxidase preparation abolished bactericidal activity which was restored on the addition of EDTA; (3) removal of trace amounts of iron by passage of salt solutions through a Chelex-100 column abolished bactericidal activity which was restored on the addition of iron.Iron and EDTA were most effective when present at 1:1 stoichiometry and they could not be replaced by a variety of other metals or chelators. The bactericidal activity of the acetaldehyde-xanthine oxidase-iron-EDTA system was inhibited by superoxide dismutase, catalase, and the OH · scavengers ethanol and mannitol, suggesting that the complex served as a catalyst of the reaction between (O

) and H₂O₂ to form OH· (Haber-Weiss reaction). Possible reasons for the relative catalytic specificity of iron-EDTA are considered.     

Effects of hypoxia and ethanol on xanthine oxidase of isolated rat hepatocytes: conversion from D to O form and leakage from cells.

The combined effects of ethanol and hypoxia on the conversion of xanthine dehydrogenase (D form) to xanthine oxidase (O form) and on the leakage of the enzyme from isolated rat hepatocytes was studied. Time-dependent death of cells occurred during incubation in hypoxic conditions. Ethanol (40 mM) had only a moderate effect on viability in aerobiosis, but accelerated the loss of hypoxic cells, which was 96% after 3 h of incubation. In hypoxic conditions, the xanthine oxidase was gradually converted from D into O form. The conversion was complete in 3 h, and was accelerated by 1 mM xanthine or by ethanol, in a concentration-related manner. Hypoxia brought about a progressive leakage of xanthine oxidase from hepatocytes, which was accelerated by ethanol in a concentration-dependent manner. The enzyme found outside hepatocytes was mostly in its O form. The xanthine oxidase of hepatocytes cytosol was converted from D into O form by human plasma or serum. In all cases the conversion could be completely reverted by treatment of the extract with dithiothreitol.     

The mechanism of liver microsomal lipid peroxidation.

In the presence of Fe-3+ and complexing anions, the peroxidation of unsaturated liver microsomal lipid in both intact microsomes and in a model system containing extracted microsomal lipid can be promoted by either NADPH and NADPH : cytochrome c reductase or by xanthine and xanthine oxidase. Erythrocuprein effectively inhibits the activity promoted by xanthine and xanthine oxidase but produces much less inhibition of NADPH-dependent peroxidation. The singlet-oxygen trapping agent, 1, 3-diphenylisobenzofuran, had no effect on NADPH-dependent peroxidation but strongly inhibited the peroxidation promoted by xanthine and xanthine oxidase. NADPH-dependent lipid peroxidation was also shown to be unaffected by hydroxyl radical scavengers.. The addition of catalase had no effect on NADPH-dependent lipid peroxidation, but it significantly increased the rate of malondialdehyde formation in the reaction promoted by xanthine and xanthine oxidase. The results demonstrate that NADPH-dependent lipid peroxidation is promoted by a reaction mechanism which does not involve either superoxide, singlet oxygen, HOOH, or the hydroxyl radical. It is concluded that NADPH-dependent lipid peroxidation is initiated by the reduction of Fe-3+ followed by the decomposition of hydroperoxides to generate alkoxyl radicals. The initiation reaction may involve some form of the perferryl ion or other metal ion species generated during oxidation of Fe-2+ by oxygen.     

Role of molecular oxygen in lignin peroxidase reactions

Homogeneous lignin peroxidase (diarylpropane oxygenase) oxidized veratryl alcohol to veratryl aldehyde under anaerobic conditions in the presence of either H2O2, m-chloroperoxybenzoic acid (mCPBA), or p-nitroperoxybenzoic acid (pNPBA). Lignin peroxidase also oxidized the 1-(3',4'-diethoxyphenyl)-1,2-dihydroxy-(4"-methoxyphenyl)-propane I under anaerobic conditions in the presence of mCPBA to yield 3,4-diethoxybenzaldehyde III and 1-(4'-methoxyphenyl)-1,2-dihydroxyethane IV. In contrast to what occurs under aerobic conditions, under anaerobic conditions no 2-hydroxy-1-(4'-methoxyphenyl)-1-oxoethane V was obtained. During the diarylpropane I cleavage under anaerobic conditions, 18O from H2(18)O was incorporated into the alpha-position of the phenylglycol IV. Lignin peroxidase also hydroxylated 1-(4'-ethoxy-3'-methoxyphenyl)propane II at the alpha-position to yield 1-(4'-ethoxy-3'-methoxyphenyl)-1-hydroxypropane VI under anaerobic conditions in the presence of mCPBA. During the phenylpropane II hydroxylation under anaerobic conditions, 18O from H2(18)O was incorporated into the alpha-position of VI. These results are rationalized according to a mechanism involving an initial one-electron oxidation of the diarylpropane I by the lignin peroxidase compound I to form a benzene pi cation radical which undergoes alpha, beta cleavage to produce a benzaldehyde and a C6C2 benzylic radical. The latter is then attacked by O2 to form a hydroperoxy radical which may decompose through a tetroxide to form the phenylglycol IV and phenylketol V. Under anaerobic conditions the C6C2 benzylic radical is probably oxidized to a carbonium ion which would be subsequently attacked by H2O to yield the phenylglycol V.     

Evidence for superoxide-dependent reduction of Fe3+ and its role in enzyme-generated hydroxyl radical formation.

This report describes studies yielding additional evidence that superoxide anion (O2) production by some biological oxidoreductase systems is a potential source of hydroxyl radical production. The phenomenon appears to be an intrinsic property of certain enzyme systems which produce superoxide and H2O2, and can result in extensive oxidative degradation of membrane lipids. Earlier studies had suggested that iron (chelated to maintain solubility) augmented production of the hydroxyl radical in such systems according to the following reaction sequence: O2 + Fe3+ leads to O2 + Fe2+ Fe2+ + H2O2 leads to Fe3+ + HO-+OH-. The data reported below provide additional support for the occurrence of these reactions, especially the reduction of Fe3+ by superoxide. Because the conditions for such reactions appear to exist in animal tissues, the results indicate a mechanism for the initiation and promotion of peroxidative attacks on membrane lipids and also suggest that the role of antioxidants in intracellular metabolism may be to inhibit initiation of degradative reactions by the highly reactive radicals formed extraneously during metabolic activity. This report presents the following new information: (1) Fe3+ is reduced to Fe2+ during xanthine oxidase activity and a significant part of the reduction was oxygen dependent. (2) Mn2+ appears to function as an efficient superoxide anion scavenger, and this function can be inhibited by EDTA. (3) The O2-dependent reduction of Fe3+ to Fe2+ by xanthine oxidase activity is inhibited by Mn2+, which, in view of statement 2 above, is a further indication that the reduction of the iron involves superoxide anion. (4) Free radical scavengers prevent or reverse the Fe3+ inhibiton of cytochrome c3+ reduction by xanthine oxidase. (5) The inhibition of xanthine oxidase-catalyzed reduction of cyt c3+ by Fe3+ does not affect uric acid production by the xanthine oxidase system. (6) The reoxidation of reduced cyt c in the xanthine oxidase system is markedly enhanced by Fe3+ and is apparently due to enhanced HO-RADICAL formation since the Fe3+-stimulated reoxidation is inhibited by free radical scavengers, including those with specificity for the hydroxyl radical.     

Carcinogenic purine N-oxide ester modifies covalently all common bases in polynucleotides

The carcinogen 1-methyl-3-hydroxyxanthine after esterification binds covalently to polynucleotides, RNA and DNA. All four ribopolynucleotides and poly(dT) are targets. Depending on reaction conditions, covalent binding is greatest to poly(A) followed by poly(U), poly(dT), poly(G), poly(C), RNA and DNA. Maximal covalent modification of DNA is one moiety per 360 nucleotides. All modified polynucleotides, RNA and DNA, except poly guanylic acid have been enzymatically digested and the major adducts characterized as nucleosides.     

Comparison of the Functional Activities of Xanthine Oxidases Isolated from Microorganisms and from Cow’s Milk

The characteristics of the formation of the superoxide radical anion (\(\rm{O}_2^{\bullet-}\)) and hydrogen peroxide by xanthine oxidases isolated from microorganisms and from cow’s milk were investigated. The increase in pH led to an increase in the rate of xanthine oxidation with oxygen by both xanthine oxidases. The functioning of xanthine oxidase from milk along with the two-electron reduction of O₂ to H₂O₂ carries through the one-electron reduction of O₂ to \(\rm{O}_2^{\bullet-}\), and the rate and the fraction of generation of \(\rm{O}_2^{\bullet-}\) increased with increasing pH. Under operation of the microbial xanthine oxidase, the \(\rm{O}_2^{\bullet-}\) radical was not detected in the medium. The results suggest a difference in the operation of active centers of enzyme from different sources.     

Formation of xanthine and the use of purine metabolites as a nitrogen source in Arabidopsis plants

In our recent paper in The Plant Journal,¹ we described the remobilization of purine metabolites during natural and dark induced senescence in wild type and Atxdh1 mutant lines impaired in xanthine dehydrogenase (XDH), a pivotal enzyme in the purine catabolism pathway. In the light of these observations and additional evidence shown here, we discuss the probable pathways leading to xanthine synthesis in Arabidopsis plants during senescence and the role that purine metabolites play as an ongoing source of nitrogen in plant growth.Key words: hypoxanthine, purine catabolism, senescence, xanthine, xanthine dehydrogenaseIn mammalian purine catabolism, hypoxanthine is oxidized to xanthine by xanthine oxidase.² In planta, xanthine can be synthesized in the purine degradation pathway, via three alternative precursors, guanine, xanthosine or hypoxanthine³ (Fig. 1A). Thus, the exact pathway leading to xanthine may depend on the species examined, the particular plant organ, developmental stage or specific environmental stimuli. For example, guanine and guanosine were shown to be the main precursor of ureides and CO₂ in cacao leaves⁴ while in tea leaves, elevated amounts of labeled xanthosine were recovered as ureides.^5,6 However, when hypoxanthine was used as a substrate for inosine monophosphate (IMP) formation in tobacco protoplasts⁷ more than 90% of labeled hypoxanthine was recovered as salvage products, nucleotides and RNA and only less then 10% was found as ureides in cacao leaves.⁴ Furthermore, when [8-¹⁴C]-hypoxanthine is supplied to soybean embryo axes or Jerusalem artichoke shoots it selectively labelled the guanine nucleotide pool.^3,8,9 These data do not support the possibility of hypoxanthine being a direct precursor for xanthine formation and illustrate the concept of species dependent differences in xanthine biosynthesis.¹⁰Open in a separate windowFigure 1Purine catabolism, xanthine and hypoxanthine accumulation and Arabidopsis plants growth. (A) Purine nucleotide catabolism in plants. Enzymes shown are: (1) AMP deaminase (EC 3.5.4.6), (2) IMP dehydrogenase (EC 1.1.1.205), (3) GMP synthase (EC 6.3.5.2), (4) 5''-nucleotidase, (5) Nucloeside phosphotransferase (EC 2.7.1.77), (6) Inosine-guanosine nucleosidase (EC 3.2.2.2), (7) Guanine deaminase (EC 3.5.4.15), (8) Xanthine dehydrogenase (EC 1.1.1.204), (9) Uricase EC 1.7.3.3, (10) Hydroxyisourate hydrolase (EC 3.5.2.17),1,18 (11) Allantoinase, allantoin amidohydrolase (EC 3.5.2.5), (12) Allantoicase, allantoate amidohydrolase (EC 3.5.3.4), (13) Ureidoglycolate lyase (EC 4.3.2.3), (14) Urease EC 3.5.1.5, (15) Allantoin deaminase (EC 3.5.3.9), (16) Ureidoglycine amidohydrolase (EC 3.5.3.-), (17) Ureidoglycolate hydrolase (EC 3.5.3.19). (B) Analysis of the purine metabolites, hypoxanthine and xanthine, in response to dark stress. Hypoxanthine and xanthine were determined by HPLC¹ in rosette leaves of wild-type (Col) and Ri14, XDH1 RNA interference plants after being kept in dark for 6 days and transferred to a 16-h light/8-h dark regime for recovery over an additional 3 days. Values are means ± SEM (n = 3). (C) Wild-type (Col) and XDH-compromised plants (KO, SALK_148364; Ri, XDH1 RNA interference) were germinated on ¼ MS medium and transplanted on the 5 day to a full MS medium (upper panel) or MS medium with 5.0 mM xanthine and urea as the sole nitrogen source. After transplanting the seedlings were left to grow for 14 days under a 16-h light/8-h dark regime (100 µmol m⁻² sec⁻¹) and then photographed. Leaf size was estimated using ImageJ software (http://rsb.info.nih.gov/ij/). Values are means ± SEM (n = 3).To study the possible role of hypoxanthine in xanthine formation in Arabidopsis we utilized XDH1 mutants. The mutants do not show any detectable XDH activity in-vitro when using hypoxanthine and/or xanthine as substrates.^1,11 Furthermore, no other enzyme is known to catalyze the conversion of hypoxanthine to xanthine, other than the molybdenum cofactor containing-XDH1. Yet, xanthine accumulation was readily detected in mutant leaves and was up to 100-fold higher than hypoxanthine either in normal growth conditions (Fig. 1B, time 0) or when exposed to dark induced senescence and to a light recovery period thereafter (Fig. 1B). These results indicate that most likely, hypoxanthine is not a major direct source for xanthine formation in Arabidopsis. The results imply that xanthosine or guanine are a source, although, one cannot exclude the possibility that hypoxanthine could be converted to xanthine in a pathway leading to inosine, IMP and then either via guanine or xanthosine, back to xanthine as illustrated in Figure 1A.In legumes inoculated with rhizobia, nitrogen is fixed initially as NH₃/NH₄⁺ that is subsequently incorporated through the purine pathway to form IMP, and finally ureides. The central role of purine catabolism in plant nitrogen metabolism was demonstrated mainly in legumes in which the purine nucleotides are degraded via uric acid and allantoin to urea and then to CO₂ and NH₃, which is then re-assimilated via the glutamine oxoglutarate aminotransferase (GOGAT) pathway (reviewed in ref. ³). What then is the role of purine catabolism pathways in non leguminous plants? Are the nitrogenous products of the degraded purines re-assimilated in non-legumes as in legume plants? We recently showed in Arabidopsis that a marked transition from assimilation, during the plants normal growth, to a state of rapid metabolite turnover occurs when plants were exposed to extended dark stress, senescence or even during normal diurnal cycles.¹⁰ This was depicted by the acceleration of purine catabolic recycling activities in which XDH1 plays a central role.¹ To test for a possible role of the accumulating purines as a source of nitrogen metabolites, we grew wild-type Arabidopsis plants and their XDH1 mutants under heterotrophic conditions. The agar plates contained either full MS nutrient solution with nitrate and ammonia or the purine metabolites, hypoxanthine (data not shown), xanthine or urea (Fig. 1C) as sole nitrogen source. The results show that the mutant plants exhibited slower growth in the medium contained xanthine or hypoxanthine compared to wild-type (Fig. 1C, lowest insert). The suboptimal growth of wild type lines is likely due to the low solubility of hypoxanthine and xanthine. In contrast, the growth on urea was the same for wild-type and XDH1 mutant transgenic plants (Fig. 1C). These results suggest that the conversion of xanthine to metabolically active intermediates, such as ureides and urea synthesized through XDH1, can play a role in ensuring nutrient supply for normal plant growth in purine containing media. Indeed, urea has been shown to be essential for the germination of Arabidopsis under nitrogenlimited conditions,¹² and recent studies have also shown that uric acid,¹³ allantoin and allantoate,^14–16 can serve as the sole nitrogen source during the growth of Arabidopsis plants. Taken together, the data suggest that ureide formation is an active component of normal plant metabolism facilitating the recovery of nitrogen in stress and non-stressed metabolism in a manner analogous to legumes. Indeed, legumes arose about 50–55 milion years ago¹⁷ and likely recruited and amplified existing plant functional purine pathways for their efficient nitrogen distribution system.     

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.