Conversion of purines to xanthine by Methanococcus vannielii

Based on the finding that Methanococcus vannielii can employ any of several purines as the sole nitrogen source, an investigation was undertaken to elucidate the pathways of purine metabolism in this organism. Cell-free extracts of M. vannielii converted guanine, uric acid, and hypoxanthine to xanthine and also formed guanine from guanine nucleotides or guanosine. The conversions of guanine and uric acid to xanthine appear to occur by pathways similar to those described in clostridia. The conversion of hypoxanthine to xanthine, however, is different than that described for Clostridium cylindrosporum and C. acidiurici, but is similar to that of C. purinolyticum, and apparently involves the direct oxidation of hypoxanthine to xanthine.     

Ferredoxin and Formyltetrahydrofolate Synthetase: Comparative Studies with Clostridium acidiurici, Clostridium cylindrosporum, and Newly Isolated Anaerobic Uric Acid-Fermenting Strains

Six strains of Clostridium acidiurici and three strains of C. cylindrosporum were isolated from soil samples by enrichment culture with uric acid as the source of carbon, nitrogen, and energy. The newly isolated strains were characterized by their spore morphology and the amounts of glycine and formate formed by the fermentation of uric acid. The strains were easily identified as belonging to one species or the other on the basis of spore morphology and formate production. The crystal properties and spectra of the native ferredoxins of all the strains isolated and the amino acid composition and partial carboxy-terminal sequence of all their apoferredoxins were determined. All the ferredoxins were tested for cross-reactivity with antiserum to C. acidiurici ferredoxin by microcomplement fixation. Five of the six C. acidiurici strains, which had ferredoxins with amino acid compositions identical to that from C. acidiurici, also showed immunological identity (immunological distance = 0.0). These results suggest sequence identity. The one strain with a different amino acid composition failed to show complete cross-reactivity. Two of the three C. cylindrosporum strains have ferredoxin amino acid compositions identical to that from C. cylindrosporum. The third strain had a minimum of five differences in sequence. All C. cylindrosporum strains had ferredoxins that differed considerably from C. acidiurici strains (minimum of eight to nine differences), and none of these ferredoxins cross-reacted with antisera to C. acidiurici ferredoxin. Antisera were prepared to formyltetrahydrofolate synthetase from C. acidiurici and C. cylindrosporum, and all possible comparisons were made by using immunodiffusion and microcomplement fixation. There is more intraspecies variation in the synthetases than in the ferredoxins; however, the results suggest considerable interspecies differences in both proteins. These results suggest a low degree of genomic relatedness between the two species, which contrasts sharply with their apparent high degree of phenotypic similarity.     

Regulation of purine hydroxylase and xanthine dehydrogenase from Clostridium purinolyticum in response to purines, selenium, and molybdenum

The discovery that two distinct enzyme catalysts, purine hydroxylase (PH) and xanthine dehydrogenase (XDH), are required for the overall conversion of hypoxanthine to uric acid by Clostridium purinolyticum was unexpected. In this reaction sequence, hypoxanthine is hydroxylated to xanthine by PH and then xanthine is hydroxylated to uric acid by XDH. PH and XDH, which contain a labile selenium cofactor in addition to a molybdenum cofactor, flavin adenine dinucleotide, and FeS centers, were purified and partially characterized as reported previously. In the present study, the activities of these two enzymes were measured in cells grown in media containing various concentrations of selenite, molybdate, and various purine substrates. The levels of PH protein in extracts were determined by immunoblot assay. The amount of PH protein, as well as the specific activities of PH and XDH, increased when either selenite or molybdate was added to the culture medium. PH levels were highest in the cells cultured in the presence of either adenine or purine. XDH activity increased dramatically in cells grown with either xanthine or uric acid. The apparent increases in protein levels and activities of PH and XDH in response to selenium, molybdenum, and purine substrates demonstrate that these enzymes are tightly regulated in response to these nutrients.     

Purification and comparative studies of dihydrolipoamide dehydrogenases from the anaerobic, glycine-utilizing bacteria Peptostreptococcus glycinophilus, Clostridium cylindrosporum, and Clostridium sporogenes.

Three different dihydrolipoamide dehydrogenases were purified to homogenity from the anaerobic glycine-utilizing bacteria Clostridium cylindrosporum, Clostridium sporogenes, and Peptostreptococcus glycinophilus, and their basic properties were determined. The enzyme isolated from P. glycinophilus showed the properties typical of dihydrolipoamide dehydrogenases: it was a dimer with a subunit molecular mass of 53,000 and contained 1 mol of flavin adenine dinucleotide and 2 redox-active sulfhydryl groups per subunit. Only NADH was active as a coenzyme for reduction of lipoamide. Spectra of the oxidized enzyme exhibited maxima at 230, 270, 353, and 453 nm, with shoulders at 370, 425, and 485 nm. The dihydrolipoamide dehydrogenases of C. cylindrosporum and C. sporogenes were very similar in their structural properties to the enzyme of P. glycinophilus except for their coenzyme specificity. The enzyme of C. cylindrosporum used NAD(H) as well as NADP(H), whereas the enzyme of C. sporogenes reacted only with NADP(H), and no reaction could be detected with NAD(H). Antibodies raised against the dihydrolipoamide dehydrogenase of C. cylindrosporum reacted with extracts of Clostridium acidiurici, Clostridium purinolyticum, and Eubacterium angustum, whereas antibodies raised against the enzymes of P. glycinophilus and C. sporogenes showed no cross-reaction with extracts from 42 organisms tested.     

Guanine uptake and metabolism in Neurospora crassa

Guanine is transported into germinated conidia of Neurospora crassa by the general purine base transport system. Guanine uptake is inhibited by adenine and hypoxanthine but not xanthine. Guanine phosphoribosyltransferase (GPRTase) activity was demonstrated in cell extracts of wild-type germinated conidia. The Km for guanine ranged from 29 to 69 micro M in GPRTase assays; the Ki for hypoxanthine was between 50 and 75 micro M. The kinetics of guanine transport differ considerably from the kinetics of GPRTase, strongly suggesting that the rate-limiting step in guanine accumulation in conidia is not that catalyzed by GPRTase. Efflux of guanine or its metabolites appears to have little importance in the regulation of pools of guanine or guanine nucleotides since very small amounts of 14C label were excreted from wild-type conidia preloaded with [8-14C]guanine. In contrast, excretion of purine bases, hypoxanthine, xanthine, and uric acid appears to be a mechanism for regulation of adenine nucleotide pools (Sabina et al., Mol. Gen. Genet. 173:31-38, 1979). No label from exogenous [8-14C]guanine was ever found in any adenine nucleotides, nucleosides, or the base, adenine, upon high-performance liquid chromatography analysis of acid extracts from germinated conidia of wild-type of xdh-l strains. The 14C label from exogenous [8-14C]guanine was found in GMP, GDP, GTP, and the GDP sugars as well as in XMP. Xanthine and uric acid were also labeled in wild-type extracts. Similar results were obtained with xdh-l extracts except that uric acid was not present. The labeled xanthine and XMP strongly suggest the presence of guanase and xanthine phosphoribosyltransferase in germinated conidia.     

Total Synthesis of Acetate from CO2 V. Determination by Mass Analysis of the Different Types of Acetate Formed from 13CO2 by Heterotrophic Bacteria

Mass analysis was used to determine the amount of acetate which is totally synthesized from (13)CO(2) during fermentations by Clostridium formicoaceticum, C. acidiurici, C. cylindrosporum, Butyribacterium rettgeri, and Diplococcus glycinophilus. In the fermentation of fructose by C. formicoaceticum, 27% of the acetate was found to be totally synthesized from CO(2), and the remaining acetate was unlabeled, having been formed from fructose. Evidence is presented that the purine-fermenting organisms, C. acidiurici and C. cylindrosporum, totally synthesized about 9% of the acetate from CO(2), and that the methyl group of an additional 9% was formed from CO(2). The remaining acetate was formed from the carbons of the purine and not via CO(2). It has been postulated that the fermentation of the purines and synthesis of acetate from CO(2) both occur via derivatives of tetrahydrofolate. Evidence is presented that a compartmentalization of these folate intermediates is required if both the purine degradation and the CO(2) utilization involve identical intermediates. Neither B. rettgeri nor D. glycinophilus incorporated sufficient (13)CO(2) into acetate to allow determination of the types of acetate by mass analysis, although they did incorporate labeled (14)CO(2) in both positions of acetate.     

Isolation of an atypically small lipoamide dehydrogenase involved in the glycine decarboxylase complex from Eubacterium acidaminophilum.

The lipoamide dehydrogenase of the glycine decarboxylase complex was purified to homogeneity (8 U/mg) from cells of the anaerobe Eubacterium acidaminophilum that were grown on glycine. In cell extracts four radioactive protein fractions labeled with D-[2-14C]riboflavin could be detected after gel filtration, one of which coeluted with lipoamide dehydrogenase activity. The molecular mass of the native enzyme could be determined by several methods to be 68 kilodaltons, and an enzyme with a molecular mass of 34.5 kilodaltons was obtained by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Immunoblot analysis of cell extracts separated by sodium dodecyl sulfate-polyacrylamide or linear polyacrylamide gel electrophoresis resulted in a single fluorescent band. NADPH instead of NADH was the preferred electron donor of this lipoamide dehydrogenase. This was also indicated by Michaelis constants of 0.085 mM for NADPH and 1.1 mM for NADH at constant lipoamide and enzyme concentrations. The enzyme exhibited no thioredoxin reductase, glutathione reductase, or mercuric reductase activity. Immunological cross-reactions were obtained with cell extracts of Clostridium cylindrosporum, Clostridium sporogenes, Clostridium sticklandii, and bacterium W6, but not with extracts of other glycine- or purine-utilizing anaerobic or aerobic bacteria, for which the lipoamide dehydrogenase has already been characterized.     

Metabolism of xanthine and hypoxanthine in the tea plant (Thea sinensis L.).

1. The metabolism of xanthine and hypoxanthine in excised shoot tips of tea was studied with micromolar amounts of [2(-14)C]xanthine or [8(-14)C]hypoxanthine. Almost all of the radioactive compounds supplied were utilized by tea shoot tips by 30 h after their uptake. 2. The main products of [2(-14)C]xanthine and [8(-14)C]hypoxanthine metabolism in tea shoots were urea, allantoin and allantoic acid. There was also incorporation of the label into theobromine, caffeine and RNA purine nucleotides. 3. The results indicate that tea plants can catabolize purine bases by the same pathways as animals. It is also suggested that tea plants have the ability to snythesize purine nucleotides from glycine by the pathways of purine biosynthesis de novo and from hypoxanthine and xanthine by the pathway of purine salvage. 4. The results of incorporation of more radioactivity from [8(-14)C]hypoxanthine than from [2(-14)C]xanthine into RNA purine nucleotides and caffeine suggest that hypoxanthine is a more effective precursor of caffeine biosynthesis than xanthine. The formation of caffeine from hypoxanthine is a result of nucleotide synthesis via the pathway of purine salvage.     

Oxidative degradation of purines by the faculative phototrophic bacterium Rhodopseudomonas capsulata

The mechanism of purine degradation was studied in the facultative phototrophic bacterium Rhodopseudomonas capsulata. Using tungstate as an inhibitor of synthesis of an active xanthine dehydrogenase it could be shown in growth experiments that purine compounds are transformed to uric acid as central purine intermediate prior to ring cleavage. Because of its rapid degradation, the mechanism of uric acid conversion was investigated using 1-methyluric acid as substrate. The analogue was partially degraded by whole cells yielding 3-methylallantoin and methylurea. This implicated an oxidative degradation of 1-methyluric acid analogous to oxidation of uric acid to allantoin suggesting uric acid degradation via allantoin. In cell-free extracts, allantoinase, allantoicase, ureidoglycolase and urease activities degrading allantoin to NH₃, CO₂ and glyoxylic acid were detected. Apparently, purine degradation in R. capsulata proceeds in a manner similar to many aerobic microorganisms. It is peculiar to this bacterium, however, that the pathway evidently operates also under anaerobic conditions. In cell extracts, oxidation of uric acid was observed which could be increased by addition of cytochrome c. The basis of this stimulation is still unknown.     

DEGRADATION OF ADENINE BY PROTOTHECA ZOPFII (CHLOROPHYTA)1

The purines 6-amino-2-hydroxypurine and 6-amino-8-hydroxypurine, not normally associated with purine degradation in algae, were isolated by high-pressure liquid chromatography from cell extracts of Prototheca zopfii Krüger grown with adenine as the sole nitrogen source .     

Uric acid synthesis by rat liver supernatants from purine bases,nucleosides and nucleotides. Effect of allopurinol

The synthesis of uric acid from purine bases, nucleosides and nucleotides has been measured in reaction mixtures containing rat liver supernatant and each one of the following compounds at 1 mM concentration (except xanthine, 0·5 mM and guanosine and guanine, 0·1 mM). The rates of the reaction, expressed as nanomoles of uric acid synthesized g^?1 of wet liver min^?1 were: ATP, 10; ADP, 37; AMP, 62; adenosine, 108; adenine 6; adenylo-succinate, 9; IMP 32; inosine, 112; hypoxanthine, 50; GTP, 19; GDP, 19; GMP, 27; guanosine, 34; guanine, 72; XMP, 10; xanthosine, 24; xanthine, 144. These figures divided by 55 correspond to nanomoles of uric acid synthesized min^?1 per mg^?1 of protein. The rate of synthesis of uric acid obtained with each one of those compounds at 0·1 and 0·05 mM concentrations was also determined. ATP (1 nM) strongly inhibited uric acid synthesis from 0·05 mM AMP (91 per cent) and from 0·05 mM ADP (88 per cent), but not from adenosine. CTP or UTP (1 mM ) also inhibited (by more than 90 per cent) the synthesis of uric acid from 0·05 mM AMP. Xanthine oxidase was inhibited by concentrations of hypoxanthine higher than 0·012 mM. The results favour the view that the level of uric acid in plasma may be an index of the energetic state of the organism. Allopurinol, besides inhibiting uric acid synthesis, reduced the rate of degradation of AMP. The ability of crude extracts to catabolize purine nucleotides to uric acid is an important factor to be considered when some enzymes related to purine nucleotide metabolism, particularly CTP synthase, are measured in crude liver extracts.     

Purine requirements for the expression of Cape buffalo serum trypanocidal activity

Cape buffalo serum contains xanthine oxidase which generates trypanocidal H₂O₂ during the catabolism of hypoxanthine and xanthine. The present studies show that xanthine oxidase-dependent trypanocidal activity in Cape buffalo serum was also elicited by purine nucleotides, nucleosides, and bases even though xanthine oxidase did not catabolize those purines. The paradox was explained in part, by the presence in serum of purine nucleoside phosphorylase and adenosine deaminase, that, together with xanthine oxidase, catabolized adenosine, inosine, hypoxanthine, and xanthine to uric acid yielding trypanocidal H₂O₂. In addition, purine catabolism by trypanosomes provided substrates for serum xanthine oxidase and was implicated in the triggering of xanthine oxidase-dependent trypanocidal activity by purines that were not directly catabolized to uric acid in Cape buffalo serum, namely guanosine, guanine, adenine monophosphate, guanosine diphosphate, adenosine 3′:5-cyclic monophosphate, and 1-methylinosine. The concentrations of guanosine and guanine that elicited xanthine oxidase-dependent trypanocidal activity were 30–270-fold lower than those of other purines requiring trypanosome-processing which suggests differential processing by the parasites.     

Uric acid synthesis by avian exocrine pancreas

1. Chicken pancreas has been shown to synthesize and secrete uric acid. Uric acid synthesis from xanthine in vitro by isolated pancreatic acinii is saturable and dependent on the activity of xanthine dehydrogenase. 2. Chicken pancreas is unable to synthesize uric acid de novo but the variety of substrates which support urate synthesis suggests that it occurs by the purine degradation pathway.     

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.     

Mesure d'une activité métabolique in vivo. Cas du catabolisme purique chez le Pharbitis nil

In vivo measurement of a metabolic activity. — Purine catabolism in Pharbitis nil cotyledons. To study the influence of light and darkness on the level of purine degradation, an in vivo method of measuring this catabolic pathway has been developed. Cotyledon discs of Pharbitis nil were incubated in an aqueous solution of hypoxanthine-8-¹⁴C. This purine base was first oxidized by a NAD⁺ dependent xanthine dehydrogenase. The metabolic flow was expressed either by the quantity of products (xanthine + uric acid + allantoin + allantoic acid) or by the rate of hypoxanthine degradation in the discs. This easy, reproducible and very sensitive technique of measurement has been investigated with respect to several variables including tissue quantity, length of incubation time, hypoxanthine and NAD⁺ concentrations, and pH. The quantity of products varied proportionally with the tissue quantity but the catabolic rate was not linked to this. The best conditions of measurement were to incubate discs for at least 60 min in distilled water containing only hypoxanthine-8-¹⁴C at low concentrations (below 0.25 mM). The values obtained represent the actual in vivo level of the studied metabolic pathway. The NAD⁺ concentration within the tissue does not seem to be a limiting factor for hypoxanthine degradation, the hypoxanthine concentration itself appears to be the only limiting factor in endogenous purine catabolism.     

Regulation of Purine Metabolism in Intact Leaves of Coffea arabica

The capacity of Coffea arabica leaves (5- x 5-mm pieces) to synthesize de novo and catabolize purine nucleotides to provide precursors for caffeine (1,3,7-trimethylxanthine) was investigated. Consistent with de novo synthesis, glycine, bicarbonate, and formate were incorporated into the purine ring of inosine 5[prime]-monophosphate (IMP) and adenine nucleotides ([sigma]Ade); azaserine, a known inhibitor of purine de novo synthesis, inhibited incorporation. Activity of the de novo pathway in C. arabica per g fresh weight of leaf tissue during a 3-h incubation period was 8 [plus or minus] 4 nmol of formate incorporated into IMP, 61 [plus or minus] 7 nmol into [sigma]Ade, and 150 nmol into caffeine (the latter during a 7-h incubation). Coffee leaves exhibited classical purine catabolism. Radiolabeled formate, inosine, adenosine, and adenine were incorporated into hypoxanthine and xanthine, which were catabolized to allantoin and urea. Urease activity was demonstrated. Per g fresh weight, coffee leaf squares incorporated 90 [plus or minus] 22 nmol of xanthine into caffeine in 7 h but degraded 102 [plus or minus] 1 nmol of xanthine to allantoin in 3 h. Feedback control of de novo purine biosynthesis was contrasted in C. arabica and Cucurbita pepo, a species that does not synthesize purine alkaloids. End-product inhibition was demonstrated to occur in both species but at different enzyme reactions.     

Purine metabolite concentrations in portal and peripheral blood of steers, sheep and rats.

1. The concentration of purine derivatives in portal and peripheral blood of steers, sheep and rats was measured by reverse-phase high performance liquid chromatography. 2. Nucleotides, nucleosides (apart from inosine), adenine and guanine were not found in the plasma samples. Allantoin, uric acid, hypoxanthine and xanthine accounted for virtually all purine metabolites in plasma samples. 3. Non-oxidized derivatives (hypoxanthine and xanthine) were consistently detected in sheep but not in steer or rat plasma samples showing a differential availability of reutilizable purine derivatives between species.     

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.     

Genetic and metabolic regulation of purine base transport in Neurospora crassa.

Summary Neurospora crassa can utilize various purine bases such as xanthine or uric acid and their catabolic products as a nitrogen source. The early purine catabolic enzymes in this organism are regulated by induction and by ammonium repression. Studies were undertaken to investigate purine base transport and its regulation in Neurospora. The results of competition experiments with uric acid and xanthine transport strongly suggest that uric acid and xanthine share a common transport system. It was also shown that the common transport system for uric acid and xanthine is distinct from a second transport system shared by hypoxanthine, adenine and guanine, and apparently also distinct from the transport system(s) for adenosine, cytosine and uracil. Regulation of the uric acid-xanthine transport system and the hypoxanthine-adenine-guanine transport system was studied. The results reveal that the uric acid-xanthine transport system is regulated by ammonium repression, but does not require uric acid induction. Neither ammonium repression nor uric acid induction controls the hypoxanthine-adenine-guanine transport system. A gene, designated amr, which is believed to be a positive regulatory gene for nitrogen metabolism of Neurospora crassa, was found to dramatically affect both the uric acid-xanthine transport system and the hypoxanthine-adenine-guanine transport system. A model for the action of the amr locus as a positive regulatory gene and for the interaction between the amr gene product and its recognition sites will be discussed.