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.     

Purification and Properties of Uricase from Enterobacter cloacae

Uricase activity was found in Enterobacter cloacae KY3074 grown on guanine, hypoxanthine, uric acid, and xanthine media. The enzyme was purified from cells grown on uric acid as a source of nitrogen. The purification procedure included ammonium sulfate fractionation, gel filtration on Sephadex G-150, and column chromatography on DEAE-cellulose and DEAE-Sephadex. The enzyme had a molecular weight of about 105,000 and was specific for uric acid. The optimum pH was around 9.5, and the activity was inhibited by the presence of potassium cyanide, Ag⁺ or Cu²⁺. This uricase can be used for estimation of uric acid.     

Trichosporon adeninovorans sp. nov., a yeast species utilizing adenine,xanthine, uric acid,putrescine and primary n-alkylamines as the sole source of carbon,nitrogen and energy

A new yeast species, Trichosporon adeninovorans, was isolated from soil by the enrichment culture method. Apart from adenine, the strain utilized uric acid, guanine, xanthine, hypoxanthine, 6,8-dihydroxypurine, putrescine, propylamine, butylamine, pentylamine, hexylamine and octylamine as sole source of carbon, nitrogen and energy.The structure of the cell wall of Tr. adeninovorans was ascomycetous. On the subcellular level growth on adenine or uric acid was accompanied with the development of microbodies in the cell. These cell organelles probably were the site of urate oxidase, an enzyme that, after growth on purine substrates, together with allantoinase was present at high activities. Low activities of adenine amidohydrolase and xanthine dehydrogenase were also demonstrated.     

Purine uptake by Alcaligenes eutrophus H16

The uptake of adenine, guanine, xanthine, hypoxanthine and uric acid by whole cells was studied, using spectrophotometric techniques, ¹⁴C-labelled compounds and metabolic inhibitors. Three different non-constitutive systems were shown to maintain the uptake of adenine and that of the pairs guanine/hypoxanthine and xanthine/uric acid. —Active transport of adenine was induced by adenine only, but passive uptake was also involved. Maximum K _T values of 110–131 M were observed at the pH optimum of 8.0. —Guanine and hypoxanthine were translocated by one single mechanism as indicated by K _T and K _I values. This system was induced by both these substances but its affinity was 51/2-times higher for guanine than for hypoxanthine; it was noncompetitively stimulated by Mg²⁺. — A further system, induced by xanthine and uric acid, catalyzed the uptake of both these compounds. It exhibited two pH optima (at pH 6.6 and 7.9); inactivation by heat and stimulation or inhibition by several compounds indicated that two separate mechanisms might be involved in the uptake of xanthine and uric acid.     

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.     

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.     

Biosynthesis of Ureides from Purines in a Cell-free System from Nodule Extracts of Cowpea [Vigna unguiculata (L) Walp.

The synthesis of ¹⁴C-labeled xanthine/hypoxanthine, uric acid, allantoin, allantoic acid, and urea from [8-¹⁴C]guanine or [8-¹⁴C]hypoxanthine, but not from [8-¹⁴C]adenine, was demonstrated in a cell-free extract from N₂-fixing nodules of cowpea (Walp.). The ¹⁴C recovered in the acid/neutral fraction was present predominantly in uric acid and allantoin (88-97%), with less than 10% of the ¹⁴C in allantoic acid and urea. Time courses of labeling in the cell-free system suggested the sequence of synthesis from guanine to be uric acid, allantoin, and allantoic acid. Ureide synthesis was confined to soluble extracts from the bacteroid-containing tissue, was stimulated by pyridine nucleotides and intermediates of the pathways of aerobic oxidation of ureides, but was completely inhibited by allopurinol, a potent inhibitor of xanthine dehydrogenase (EC 1.2.1.37). The data indicated a purine-based pathway for ureide synthesis by cowpea nodules, and this suggestion is discussed.     

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

1. Absorption of purines and their metabolism by the small intestine were estimated by using the everted gut sacs from the duodenum, jejunum and ileum of the chicken. 2. When no purine was added to the mucosal fluid, large amounts of uric acid, much less but appreciable adenine, hypoxanthine and xanthine and no detectable guanine were released from both sides of all segments of the small intestine, and these released amounts were largest in the duodenum. 3. Similar absorption rates of adenine from the jejunum and ileum were about 1.7-3.0 times as high as those of hypoxanthine and uric acid from these intestines and those of adenine and uric acid from the duodenum (P less than 0.05). 4. Guanine was not absorbed unchanged from any segments of the intestine and a little xanthine was absorbed only from the jejunum and ileum. 5. Guanine and xanthine seem to be absorbed in uric acid form, hypoxanthine in xanthine and uric acid forms and adenine in hypoxanthine form, from the small intestine especially from the jejunum. 6. Adenine, guanine, xanthine and hypoxanthine were greatly metabolized in the mucosa of the duodenum, and the conversions of hypoxanthine to xanthine and uric acid were most active.     

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.     

Petroleum Bioremediation in Seawater Using Guano as the Fertilizer

The biodegradation of hydrocarbon pollutants in open systems, such as oceans, is generally limited by the availability of utilizable nitrogen and phosphorus sources. Here the authors demonstrate the potential of overcoming this problem with guano as the fertilizer. In the first set of experiments, the principle and conditions for growing bacteria on a water insoluble fertilizer was established, using uric acid as the nitrogen source and a pure culture of an isolated hydrocarbon-degrading bacterium, Alcanivorax sp. OK2. Using a simulated open system, it was demonstrated that uric acid (the major nitrogen component of guano) binds to crude oil and is available for the growth of strain OK2 and petroleum degradation. In the second set of experiments, using a simulated open system, it was demonstrated that commercial guano was an effective source of nitrogen and phosphorus for the growth of marine bacteria on crude oil. Bacterial cultures reached over 10⁸ cells per ml and 70% of the crude oil was degraded. Controls using ammonium sulfate and phosphate in place of guano in the simulated open system reached only 10⁶ cells per ml and showed no detectable hydrocarbon degradation. Isolation and characterization of the bacteria in the crude oil/guano cultures indicated that they were primarily strains of Alcanivorax and Alteromonas.     

Identification of xanthine and hypoxanthine as components of assembly pheromone in excreta of argasid ticks

In addition to guanine, xanthine and hypoxanthine were identified in white spherules in excreta of five species ofArgas andOrnithodoros ticks by a reverse-phase high-performance liquid chromatography (HPLC) and a gas chromatographic method with mass spectrometric detection, (GC/MS). The mutual relationships of these purines in excreta ofArgas (Persicargas) persicus were found to be less than 1.5% for hypoxanthine, less than 9.0% for xanthine and 89.8–98.6%, for guanine. In excreta of other species, the relationships of purines were similar, with the exception ofArgas (A.) reflexus andA. (A.) polonicus, where the amount of hypoxanthine was rather elevated. Uric acid was also identified in some cases. The assembly efficacy of xanthine and hypoxanthine is similar to that of guanine, but xanthine significantly enhances the assembly efficacy of commercial guanine when mixed in ratio of about 125. Thus, xanthine seems to be the second important component of assembly pheromone of argasid ticks.     

The association between serum ferritin and uric acid in humans

Objective: Urate forms a coordination complex with Fe³⁺ which does not support electron transport. The only enzymatic source of urate is xanthine oxidoreductase. If a major purpose of xanthine oxidoreductase is the production of urate to function as an iron chelator and antioxidant, a system for coupling the activity of this enzyme to the availability of catalytically-active metal would be required. We tested the hypothesis that there is an association between iron availability and urate production in healthy humans by correlating serum concentrations of ferritin with uric acid levels.

Materials and methods: The study population included 4932 females and 4794 males in the National Health and Nutrition Examination Survey III. They were 20 years of age or older and in good health.

Results: Serum concentrations of ferritin correlated positively with uric acid levels in healthy individuals (R²=0.41, p<0.001). This association was independent of an effect of gender, age, race/ethnic group, body mass, and alcohol consumption.

Conclusions: The relationship between serum ferritin and uric acid predicts hyperuricemia and gout in groups with iron accumulation. This elevation in the production of uric acid with increased concentrations of iron could possibly reflect a response of the host to diminish the oxidative stress presented by available metal as the uric acid assumes the empty or loosely bound coordination sites of the iron to diminish electron transport and subsequent oxidant generation.     

Nitrogen Metabolism in Paramecium aurelia

Nitrogen in cell fractions of Paramecium aurelia varied according to the growth medium. Trichloroacetic acid-soluble fractions of cells were chromatographer. Adenine, adenosine, guanine, guanosine, hypoxanthine, aspartic acid, glutamic acid, histidine, lysine, proline, and phenylalanine were identified. Fyrimidines and xanthine, or their respective ribosides and ribotides, were not detected. Ammonia was released into the medium by both actively growing and "resting" cells. Culture fluids of "resting"cells also contained hypoxanthine and lesser amounts of adenine and guanine. Urea, uric acid, creatine, cretonne, and ailantoin were absent.
Pyrimidine nitrogen seems excreted as dihydrouracil. The following enzymes were detected in homogenates and cell-free preparations: nucleotidases, nucleoside hydrolases, and cytidine deaminase. Urease, uricase, adenase, guanase, xanthine oxidase, adenosine deaminase, and 5'-adenylic acid deaminase were not present in this organism.
Purine and pyrimidine incorporation into nucleic acids was investigated by the use of radioactive tracers. Guanosine gives rise to nucleic-acid guanine and adenine; adenosine was precursor to nucleic acid adenine only. Formate was incorporated into purines; glycine was not. P. aurelia can interconvert cytidine and uridine; both give rise to nucleic acid thymine. The methyl group of thymine may be derived from formate.     

Purine Composition of the Crystalline Cytoplasmic Inclusions of Paramecium tetraurelia

Crystalline cytoplasmic inclusions were isolated by differential centrifugation from mass cultures of Paramecium tetraurelia feeding on Klebsiella pneumonia. Physical and chemical measurements of intact and solubilized crystals determined that they consist primarily of guanine and hypoxanthine with traces of xanthine. Crystals from the mutant sombre consist primarily of xanthine, suggesting there is a disorder of purine metabolism in this mutant.     

Effect of allopurinol on the utilization of purine degradation pathway intermediates by tobacco cell cultures

Suspension cultured Nicotiana tabacum (tobacco) cells grow slowly on intermediates of the purine degradation pathway (hypoxanthine, xanthine, uric acid, allantoin, and urea) as their sole nitrogen source indicating that this degradation pathway is operative in these cells. The hypoxanthine analog, allopurinol inhibited tobacco cell growth on hypoxanthine but not uric acid. This helps confirm that the site of action of allopurinol is the conversion of hypoxanthine to uric acid by xanthine oxidase. Attempts to select cells which could grow in the presence of allopurinol with hypoxanthine as the nitrogen source were not successful.     

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.     

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.     

Degradation of Uric Acid by Certain Aerobic Bacteria

We have isolated and identified nine cultures of aerobic bacteria capable of growing on an elective medium containing uric acid as the only source of carbon, nitrogen, and energy. Four of these cultures were identified as Aerobacter aerogenes, two as Klebsiella pneumoniae, and the remainder as Serratia killiensis, Pseudomonas aeruginosa, and Bacillus species. Another culture identified as P. fluorescens required both glucose and uric acid for growth. When 23 laboratory stock cultures were inoculated into the uric acid medium, A. aerogenes, B. subtilis, Mycobacterium phlei, P. aeruginosa, and S. marcescens were able to grow. These five cultures also grew when the uric acid was replaced with adenine, guanine, hypoxanthine, xanthine, or allantoin, but growth was poor. In all of these media, including the uric acid medium, addition of glucose along with the nitrogenous compounds yielded good growth. Induction experiments demonstrated that the ability of A. aerogenes, K. pneumoniae, P. aeruginosa, P. fluorescens, S. kiliensis, S. marcescens, B. subtilis, and Bacillus sp. to degrade uric acid is an induced property. Of these organisms, only Bacillus sp. accumulated a small amount of intracellular uric acid.     

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

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

Utilization of organic compounds as the sole source of nitrogen by Thiobacillus thiooxidans

Thiobacillus thiooxidans DSM 504 was shown to grow with adenine, hypoxanthine, xanthine and uric acid as sole sources of nitrogen. Growth with these compounds was observed after lag periods of varying lengths, unless the cells had been previously grown with the same purine base. The disappearance of adenine was accompanied by a temporary accumulation of hypoxanthine in the medium. The utilization of purines was inhibited by ammonia (1 mM). Guanine, pyrimidines and some other organic compounds were not utilized.Non-standard abbreviation U-¹⁴C uniformly labeled by ¹⁴C