Effect of dietary protein on purine nucleoside phosphorylase and xanthine dehydrogenase activities of liver and kidney in chicken and pigeon

Comparative studies were made on the effects of diets of different protein contents on the activities of purine nucleoside phosphorylase and xanthine dehydrogenase of avian livers and kidneys. In chicken liver and kidney, both enzyme activities were increased with high protein diet, confirming the previous results. In pigeon liver, only purine nucleoside phosphorylase was increased but xanthine dehydrogenase activity was not detected after feeding a high protein diet, while both enzyme activities were increased in the pigeon kidney. The increase in the levels of plasma oxypurines in pigeon serum was consistent with the result that the xanthine dehydrogenase activity of pigeon was not detected in the liver but in the kidney.     

Regulation of nitrogen catabolic enzymes in chick liver: effects of insulin.

Previous studies have shown that a group of nitrogen catabolic enzymes including xanthine dehydrogenase, purine nucleoside phosphorylase, and tyrosine aminotransferase are all increased in chick liver by dietary protein as well as single amino acids (e.g. methionine) and certain antimetabolites (e.g. hydrazine). A similar enzyme response pattern can be obtained with insulin. This hormone causes an enhanced rate of XDH synthesis and gives nonadditive results with protein, hydrazine and methionine. Furthermore, a vitamin B6 dependency was observed in responses to both high protein diets and insulin, all suggesting a common regulatory mechanism. In this system dietary protein and insulin may act similarly by increasing the availability of amino acids to the liver -- in one case by supplying amino acids through the diet and in the other by increasing amino acid uptake.     

Inosine synthesis and phosphoribosylpyrophosphate availability in rat liver cells in the presence of allopurinol

In the presence of allopurinol, apparent phosphoribosylpyrophosphate (PP-ribose-P) availability as measured by adenine incorporation into ribonucleotides was decreased in rat liver cells, hypoxanthine incorporation into ribonucleotides was increased, and there was a large synthesis of inosine from hypoxanthine. Inosine was formed directly by the reversal of the purine nucleoside phosphorylase reaction which was very rapid in liver cells. We tested the hypothesis that utilization of ribose 1-phosphate for inosine synthesis could decrease PP-ribose-P availability. Our results indicate that the apparent decrease of PP-ribose-P availability in the presence of allopurinol was due to competition between adenine and hypoxanthine salvage pathways into nucleotides, and not to the synthesis of inosine.     

Regulation of xanthine dehydrogenase in chick liver. Effect of starvation and of administration of purines and purine nucleosides

1. The xanthine-dehydrogenase activity of chick liver, expressed per mg. of nitrogen, is increased during starvation. 2. Administration of inosine and possibly of adenine has a comparable effect on the xanthine dehydrogenase, and also induces an elevation of the total quantity of enzyme. Hypoxanthine, xanthine, guanine, xanthosine, guanosine and adenosine are ineffective. Cortisone is equally ineffective. 3. The administration of puromycin abolishes the effect of inosine and reduces that of starvation. It is concluded that inosine induces an increased synthesis of xanthine dehydrogenase, whereas during starvation the enzyme is spared with respect to other liver proteins. 4. The hypothesis is formulated that chick-liver xanthine dehydrogenase is an adaptive enzyme, its activity being regulated by inosine or by one of its metabolites.     

Profiles of purine biosynthesis, salvage and degradation in disks of potato (Solanum tuberosum L.) tubers

To find general metabolic profiles of purine ribo- and deoxyribonucleotides in potato (Solanum tuberosum L.) plants, we looked at the in situ metabolic fate of various ¹⁴C-labelled precursors in disks from growing potato tubers. The activities of key enzymes in potato tuber extracts were also studied. Of the precursors for the intermediates in de novo purine biosynthesis, [¹⁴C]formate, [2-¹⁴C]glycine and [2-¹⁴C]5-aminoimidazole-4-carboxyamide ribonucleoside were metabolised to purine nucleotides and were incorporated into nucleic acids. The rates of uptake of purine ribo- and deoxyribonucleosides by the disks were in the following order: deoxyadenosine > adenosine > adenine > guanine > guanosine > deoxyguanosine > inosine > hypoxanthine > xanthine > xanthosine. The purine ribonucleosides, adenosine and guanosine, were salvaged exclusively to nucleotides, by adenosine kinase (EC 2.7.1.20) and inosine/guanosine kinase (EC 2.7.1.73) and non-specific nucleoside phosphotransferase (EC 2.7.1.77). Inosine was also salvaged by inosine/guanosine kinase, but to a lesser extent. In contrast, no xanthosine was salvaged. Deoxyadenosine and deoxyguanosine, was efficiently salvaged by deoxyadenosine kinase (EC 2.7.1.76) and deoxyguanosine kinase (EC 2.7.1.113) and/or non-specific nucleoside phosphotransferase (EC 2.7.1.77). Of the purine bases, adenine, guanine and hypoxanthine but not xanthine were salvaged for nucleotide synthesis. Since purine nucleoside phosphorylase (EC 2.4.2.1) activity was not detected, adenine phosphoribosyltransferase (EC 2.4.2.7) and hypoxanthine/guanine phosphoribosyltransferase (EC 2.4.2.8) seem to play the major role in salvage of adenine, guanine and hypoxanthine. Xanthine was catabolised by the oxidative purine degradation pathway via allantoin. Activity of the purine-metabolising enzymes observed in other organisms, such as purine nucleoside phosphorylase (EC 2.4.2.1), xanthine phosphoribosyltransferase (EC 2.4.2.22), adenine deaminase (EC 3.5.4.2), adenosine deaminase (EC 3.5.4.4) and guanine deaminase (EC 3.5.4.3), were not detected in potato tuber extracts. These results suggest that the major catabolic pathways of adenine and guanine nucleotides are AMP → IMP → inosine → hypoxanthine → xanthine and GMP → guanosine → xanthosine → xanthine pathways, respectively. Catabolites before xanthosine and xanthine can be utilised in salvage pathways for nucleotide biosynthesis.     

Purine and oxypurine production in mitochondria of ischemic and reperfused myocardium

The present study was undertaken to determine whether significant breakdown of adenine nucleotides to purine bases and oxypurines occurred in mitochondria following myocardial ischemia and ischemia followed by reperfusion, and whether allopurinol prevented this effect. The adenine nucleotides adenosine, hypoxanthine, xanthine and uric acid were measured in the mitochondria and the results suggest that breakdown did occur. Malondialdehyde concentration was determined to gauge lipid peroxidation. This substance did not increase during ischemia or reperfusion, but did so in the presence of allopurinol. Xanthine dehydrogenase was converted to xanthine oxidase during reperfusion and the activity of both enzymes were inhibited by allopurinol. The results also suggested the presence of a mitochondrial 5'-nucleotidase. We conclude that significant breakdown of adenine nucleotide took place in myocardial mitochondria during ischemia and ischemia followed by reperfusion and that allopurinol may have a protective effect.     

Characteristics of purine nucleotide metabolism and GMP-reductase activity in chicken tissues

The [3H]guanosine and [3H]guanine label is shown to be distributed unevenly in the purine components of chicken tissues. 60 min after isotope administration about 80% of radioactivity is localized in xanthine and uric acid in the liver and duodenum, that agrees with high activity of purine nucleoside phosphorylase (EC 2.4.2.1) and guanine deaminase (EC 3.5.4.3). At the same time over 50% of label is found in the spleen in adenine nucleotides of the pool, RNA as well as in hypoxanthine and only 20% in oxypurines. Such a distribution of the label is in direct correlation with the activity of GMP-reductase (EC 1.6.6.8) catalyzing the reduction deamination of GMP in IMP.     

Regulation of some nitrogen catabolizing enzyme levels in chick liver.

Xanthine dehydrogenase, purine nucleoside phosphorylase, and tyrosine aminotransferase are all increased sharply in liver of chicks by dietary protein. Results in this paper show that most amino acids have this effect, with methionine and tryptophan being more effective than the remainder. This suggested that any source of amino nitrogen could cause a build-up of a nitrogenous intermedate(s) necessary for enzyme accumulation. The above hypothesis was tested by blocking the breakdown of amino acids using various antimetabolites. Results were uniformly successful in raising enzyme levels, and furthermore it was found that antimetabolites and amino acids are not additive, suggesting that they act by a similar mechanism.     

Suppression of chick pancreatic xanthine dehydrogenase by unnsaturated fatty acids

A study of pancreatic xanthine dehydrogenase levels in chicks immediately after hatching has revealed that the sharp developmental increase normally occurring can be suppressed by maintenance on whole egg (Fisheret al., 1967; Woodward and Fisher, 1967). Further work has shown that ether extracts of egg yolks are equally effective in supressing this developmental increase. Of the lipid components present, four have been found to affect the accumulation of pancreatic xanthine dehydrogenase: linolenic, linoleic, and oleic acids suppress and stearic acid enhances. It seems reasonable to believe that the suppressing activity of ether extracts of eggs is due to the presence of unsaturated fatty acids. The administration of specific fatty acids leads to increased concentrations of these materials in the lipids of the pancreas.     

Metabolism of Guanine and Guanine Nucleotides in Primary Rat Neuronal Cultures

The metabolic fate of guanine and of guanine ribonucleotides (GuRNs) in cultured rat neurons was studied using labeled guanine. 8-Aminoguanosine (8-AGuo), an inhibitor of purine nucleoside phosphorylase, was used to clarify the pathways of GMP degradation, and mycophenolic acid, an inhibitor of IMP dehydrogenase, was used to assess the flux from IMP to GMP and, indirectly, the activity of the guanine nucleotide cycle (GMP----IMP----XMP----GMP). The main metabolic fate of guanine in the neurons was deamination to xanthine, but significant incorporation of guanine into GuRNs, at a rate of approximately 8.5-13.1% of that of the deamination, was also demonstrated. The turnover rate of GuRNs was fast (loss of 80% of the radioactivity of the prelabeled pool in 22 h), reflecting synthesis of nucleic acids (32.8% of the loss in radioactivity) and degradation to xanthine, guanine, hypoxanthine, guanosine, and inosine (49.3, 4.3, 4.1, 1.1, and 0.5% of the loss, respectively). Of the radioactivity in GuRNs, 7.9% was shifted to adenine nucleotides. The accumulation of label in xanthine indicates (in the absence of xanthine oxidase) that the main degradative pathway from GMP is that to xanthine through guanosine and guanine. The use of 8-AGuo confirmed this pathway but indicated the operation of an additional, relatively slower degradative pathway, that from GMP through IMP to inosine and hypoxanthine. Hypoxanthine was incorporated mainly into adenine nucleotide (91.5%), but a significant proportion (6%) was found in GuRNs.(ABSTRACT TRUNCATED AT 250 WORDS)     

Transport of adenine, hypoxanthine and uracil into Escherichia coli.

Uptake of adenine, hypoxanthine and uracil by an uncA strain of Escherichia coli is inhibited by uncouplers or when phosphate in the medium is replaced by less than 1 mM-arsenate, indicating a need for both a protonmotive force and phosphorylated metabolites. The rate of uptake of adenine or hypoxanthine was not markedly affected by a genetic deficiency of purine nucleoside phosphorylase. In two mutants with undetected adenine phosphoribosyltransferase, the rate of adenine uptake was about 30% of that in their parent strain, and evidence was obtained to confirm that adenine had then been utilized via purine nucleoside phosphorylase. In a strain deficient in both enzymes adenine uptake was about 1% of that shown by wild-type strains. Uptake of hypoxanthine was similarly limited in a strain lacking purine nucleoside phosphorylase, hypoxanthine phosphoribosyltransferase and guanine phosphoribosyltransferase. Deficiency of uracil phosphoribosyltransferase severely limits uracil uptake, but the defect can be circumvented by addition of inosine, which presumably provides ribose 1-phosphate for reversal of uridine phosphorylase. The results indicate that there are porter systems for adenine, hypoxanthine and uracil dependent on a protonmotive force and facilitated by intracellular metabolism of the free bases.     

Development of a FIA system with immobilized enzymes for specific post-column detection of purine bases and their nucleosides separated by HPLC column.

A sensitive and highly selective method for the simultaneous determination of purine bases and their nucleosides is proposed. An amperometric flow-injection system with the two immobilized enzyme reactors (guanase immobilized reactor and purine nucleoside phosphorylase/xanthine oxidase co-immobilized reactor) is used as the specific post-column detection system of HPLC, to convert compounds separated by a reversed-phase. HPLC column to electroactive species (hydrogen peroxide and uric acid) which can be detected at a flow-through platinum electrode. The proposed detection system is specific for a group of purine bases and purine nucleosides and does not respond for purine nucleotides and pyrimidine bases. The linear determination ranges are from 10 pmol to 5 nmol for four purine bases (hypoxanthine, xanthine, guanine, and adenine) and four purine nucleosides (inosine, xanthosine, guanosine, and adenosine). The detection limits are 1.2-5.5 pmol.     

Phosphorylase-mediated mobilization of the amino group of adenine in Bacillus cereus

Mobilization of the ribose moiety of purine nucleosides as well as of the amino group of adenine may be realized in Bacillus cereus by the concerted action of three enzymes: adenosine phosphorylase, adenosine deaminase, and purine nucleoside phosphorylase. In this pathway, ribose-1-phosphate and inorganic phosphate act catalytically, being continuously regenerated by purine nucleoside phosphorylase and adenosine phosphorylase, respectively. As a result of such a metabolic pathway, adenine is quantitatively converted into hypoxanthine, thus overcoming the lack of adenase in B. cereus.     

A quantitative histochemical procedure for the demonstration of purine nucleoside phosphorylase activity in rat and human liver using Tetranitro BT and xanthine oxidase as auxiliary enzyme

Summary A quantitative histochemical procedure was developed for the demonstration of purine nucleoside phosphorylase in rat liver using unfixed cryostat sections and the auxiliary enzyme xanthine oxidase. The optimum incubation medium contained 18% (w/v) poly(vinyl alcohol), 100 mM phosphate buffer, pH 8.0, 0.5 mm inosine, 0.47 mm methoxyphenazine methosulphate and 1 mm Tetranitro BT. An enzyme film consisting of xanthine oxidase was brought onto the object slides before the section was allowed to adhere. The specificity of the reaction was proven by the low amount of final reaction product generated when incubating in the absence of inosine. Moreover, 1 mm p-chloromercuribenzoic acid, a non-specific inhibitor of purine nucleoside phosphorylase, inhibited the specific reaction by 90%. The specific reaction defined as the test reaction, in the presence of substrate, minus the control reaction, in the absence of substrate was linear with incubation time at least up to 30 min as measured cytophotometrically. A high activity was observed in endothelial cells and Kupffer cells of rat liver and a lower activity in liver parenchymal cells. Pericentral hepatocytes showed an activity higher than that of periportal hepatocytes. In human liver, purine nucleoside phosphorylase activity was also high in endothelial cells and Kupffer cells, but the activity in liver parenchymal cells was only slightly lower than it was in non-parenchymal cells. The localization of the enzyme is in agreement with earlier ultrastructural findings using fixed liver tissue and the lead salt procedure.     

Cloning,heterologous expression,and in situ characterization of the first high affinity nucleobase transporter from a protozoan

While multiple nucleoside transporters, some of which can also transport nucleobases, have been cloned in recent years from many different organisms, no sequence information is available for the high affinity, nucleobase-selective transporters of metazoa, parazoa, or protozoa. We have identified a gene, TbNBT1, from Trypanosoma brucei brucei that encodes a 435-residue protein of the equilibrative nucleoside transporter superfamily. The gene was expressed in both the procyclic and bloodstream forms of the organism. Expression of TbNBT1 in a Saccharomyces cerevisiae strain lacking an endogenous purine transporter allowed growth on adenine as sole purine source and introduced a high affinity transport activity for adenine and hypoxanthine, with Km values of 2.1 +/- 0.6 and 0.66 +/- 0.22 microm, respectively, as well as high affinity for xanthine, guanine, guanosine, and allopurinol and moderate affinity for inosine. A transporter with an indistinguishable kinetic profile was identified in T. b. brucei procyclics and designated H4. RNA interference of TbNBT1 in procyclics reduced cognate mRNA levels by approximately 80% and H4 transport activity by approximately 90%. Expression of TbNBT1 in Xenopus oocytes further confirmed that this gene encodes the first high affinity nucleobase transporter from protozoa or animals to be identified at the molecular level.     

The site of histone H2b phosphorylated by a cyclic nucleotide independent histone kinase

Reserpine, a Rauwolfia alkaloid, was shown to increase activity of the hepatic nitrogen metabolizing enzymes xanthine dehydrogenase, purine nucleoside phosphorylase, and tyrosine aminotransferase, when administered orally to young chicks. Using immunochemical techniques, this increase in xanthine dehydrogenase was shown to result from an enhanced de novo enzyme synthesis. The response pattern of the three enzymes to reserpine follows the same pattern to induction by high dietary protein suggesting that a common mode of action may be involved in the regulation of these enzymes. Alpha-Adrenergic blockers, phentolamine and phenoxybenzamine, effectively prevented the increased enzyme activities caused by administration of reserpine.     

Genetic and physiological characterization of the purine salvage pathway in the archaebacterium Methanobacterium thermoautotrophicum Marburg.

The enzymes involved in the purine interconversion pathway of wild-type and purine analog-resistant strains of Methanobacterium thermoautotrophicum Marburg were assayed by radiometric and spectrophotometric methods. Wild-type cells incorporated labeled adenine, guanine, and hypoxanthine, whereas mutant strains varied in their ability to incorporate these bases. Adenine, guanine, hypoxanthine, and xanthine were activated by phosphoribosyltransferase activities present in wild-type cell extracts. Some mutant strains simultaneously lost the ability to convert both guanine and hypoxanthine to the respective nucleotide, suggesting that the same enzyme activates both bases. Adenosine, guanosine, and inosine phosphorylase activities were detected for the conversion of base to nucleoside. Adenine deaminase activity was detected at low levels. Guanine deaminase activity was not detected. Nucleoside kinase activities for the conversion of adenosine, guanosine, and inosine to the respective nucleotides were detected by a new assay. The nucleotide-interconverting enzymes AMP deaminase, succinyl-AMP synthetase, succinyl-AMP lyase, IMP dehydrogenase, and GMP synthetase were present in extracts; GMP reductase was not detected. The results indicate that this autotrophic methanogen has a complex system for the utilization of exogenous purines.     

Mutants of Escherichia coli Defective in Ribonucleoside and Deoxyribonucleoside Catabolism

From Escherichia coli B, mutants were prepared that lacked the enzymes adenosine deaminase, cytidine deaminase, and purine nucleoside phosphorylase. In each case, the mutant lacked enzyme activity for both ribonucleoside and deoxyribonucleoside. Mutants lacking purine nucleoside phosphorylase lost the capacity to cleave the nucleosides of adenine, guanine, and hypoxanthine.     

Metabolism of Exogenous Purine Bases and Nucleosides by Salmonella typhimurium

Purine-requiring mutants of Salmonella typhimurium LT2 containing additional mutations in either adenosine deaminase or purine nucleoside phosphorylase have been constructed. From studies of the ability of these mutants to utilize different purine compounds as the sole source of purines, the following conclusions may be drawn. (i) S. typhimurium does not contain physiologically significant amounts of adenine deaminase and adenosine kinase activities. (ii) The presence of inosine and guanosine kinase activities in vivo was established, although the former activity appears to be of minor significance for inosine metabolism. (iii) The utilization of exogenous purine deoxyribonucleosides is entirely dependent on a functional purine nucleoside phosphorylase. (iv) The pathway by which exogenous adenine is converted to guanine nucleotides in the presence of histidine requires a functional purine nucleoside phosphorylase. Evidence is presented that this pathway involves the conversion of adenine to adenosine, followed by deamination to inosine and subsequent phosphorolysis to hypoxanthine. Hypoxanthine is then converted to inosine monophosphate by inosine monophosphate pyrophosphorylase. The rate-limiting step in this pathway is the synthesis of adenosine from adenine due to lack of endogenous ribose-l-phosphate.     

Induction and repression of enzymes involved in exogenous purine compound utilization of Bacillus cereus

5'-Nucleotidase, adenosine phosphorylase, adenosine deaminase and purine nucleoside phosphorylase, four enzymes involved in the utilization of exogenous compounds in Bacillus cereus, were measured in extracts of this organism grown in different conditions. It was found that adenosine deaminase is inducible by addition of adenine derivatives to the growth medium, and purine, nucleoside phosphorylase by metabolizable purine and pyrimidine ribonucleosides. Adenosine deaminase is repressed by inosine, while both enzymes are repressed by glucose. Evidence is presented that during growth of B. cereus in the presence of AMP, the concerted action of 5'-nucleotidase and adenosine phosphorylase, two constitutive enzymes, leads to formation of adenine, and thereby to induction of adenosine deaminase. The ionsine formed would then cause induction of the purine nucleoside phosphorylase and repression of the deaminase. Taken together with our previous findings showing that purine nucleoside phosphorylase of B. cereus acts as a translocase of the ribose moiety of inosine inside the cell (Mura, U., Sgarrella, F. and Ipata, P.L. (1978) J. Biol Chem. 253, 7905-7909), our results provide a clear picture of the molecular events leading to the utilization of the sugar moiety of exogenous AMP, adenosine and inosine as an energy source.