Purine Ribonucleotide Synthesis Research Articles

A Prebiotic Synthesis of Canonical Pyrimidine and Purine Ribonucleotides.

The “RNA first” model for the origin of life holds that RNA emerged spontaneously on early Earth and developed into life through its dual capabilities for genetics and catalysis. The model's central weakness is the difficulty of making its building blocks, in particular, the glycosidic bond joining nucleobases to ribose. Thus, the focus of much of the modern literature on the topic is directed toward solving this difficulty and includes elegant, though indirect, methods for making this bond. Here, we report that the glycosidic bond in canonical pyrimidine and purine ribonucleotides can be formed by direct coupling of cyclic carbohydrate phosphates with free nucleobases, all reported to be available by experimentally supported pathways that might have operated on early Earth.

Methotrexate inhibits the first committed step of purine biosynthesis in mitogen-stimulated human T-lymphocytes: a metabolic basis for efficacy in rheumatoid arthritis?

The immunosuppressive and anti-inflammatory effects of low-dose methotrexate (MTX) have been related directly to inhibition of folate-dependent enzymes by polyglutamated derivatives, or indirectly to adenosine release and/or apoptosis and clonal deletion of activated peripheral blood lymphocytes in S-phase. In this study of phytohaemagglutinin-stimulated primary human T-lymphocytes we show that MTX (20 nM to 20 microM) was cytostatic not cytotoxic, halting proliferation at G(1). This stasis of blastogenesis was associated with an inhibition of purine ribonucleotide synthesis but a stimulation of pyrimidine biosynthesis, the normal mitogen-induced expansion of ATP and GTP pools over 72 h being restricted to concentrations of unstimulated T-cells, whereas the increment in UTP pools exceeded that of controls. Decreased incorporation of H(14)CO(3) or [(14)C]glycine into purine ribonucleotides, with no radiolabel accumulation in any de novo synthetic intermediate but enhanced H(14)CO(3) incorporation into UTP, supported these MTX-related effects. Exaggerated [(14)C]hypoxanthine salvage (which normalized the purine and UTP pools) confirmed the increased availability of 5-phosphoribosyl-1-pyrophosphate (PP-ribose-P) as the molecular mechanism underlying these disparate changes. These results provide the first substantive evidence that the immunosuppressive effects of low-dose MTX in primary blasting human T-lymphocytes relate not to the inhibition of the two folate-dependent enzymes of purine biosynthesis but to inhibition of the first enzyme, amidophosphoribosyltransferase, thereby elevating PP-ribose-P and stimulating UTP synthesis. Varying cell types or incubation conditions employed by other workers, especially malignant/activated cells with high basal metabolic rates, might mask the effects noted in primary human T-lymphocytes. The findings imply the involvement of low-dose MTX in the inhibition of T-lymphocyte proliferation and proliferation-dependent processes in rheumatoid arthritis.

Increased de novo purine synthesis by insulin through selective enzyme induction in primary cultured rat hepatocytes

The proliferative effect of insulin on de novo purine synthesis and on the expression of various enzymes of purine metabolism were studied in primary cultured rat hepatocytes. Insulin greater than 1.5 x 10(-8) M increased DNA and de novo purine synthesis to 260-390 and 270-420%, respectively, 24 and 8 h after the administration. Insulin at 1.5 x 10(-7) M increased the specific activity of amidophosphoribosyltransferase (ATase) to 154-180%, hypoxanthine-guanine phosphoribosyltransferase to 129%, and adenine phosphoribosyltransferase (APRT) to 205%, in contrast to unchanged xanthine dehydrogenase at 80%. Enzyme induction was supported by the results of kinetic analysis and the inhibition of the insulin-induced increase in enzyme activities by protein synthesis inhibitors. Insulin increased ATP to 127% and decreased AMP, ADP, 5'-guanylic acid (GMP), and guanosine 5'-diphosphate (GDP), respectively, to 73, 69, 73, and 69%. Insulin increased adenylate energy charge from 0.83 to 0.90 without changing total feedback inhibitory potential on ATase. No obvious increase of 5-phosphoribosyl-1-pyrophosphate supply was suggested, although its apparent availability for purine ribonucleotide synthesis was increased to 208-245%, reflecting mainly induced APRT activity to 205%. It is concluded that hepatocyte proliferation by insulin, as evidenced by purine metabolism, is mediated by the selective gene activation of anabolic enzymes and increased ATP as the basis to activate multiple metabolic pathways without remarkable changes of substrate availability or feedback inhibition.

Cobalamin and folate metabolism in helminths

Knowledge about the source, metabolism and functions of cobalamins and folates in helminths remains fragmentary. It is likely that all helminths, whether free-living or parasitic, cannot synthesize cobalamins and folates de novo. Folates, but not cobalamins, appear to be ubiquitous in helminths. Of the parasitic helminths that take up free cobalamins in vitro, all but one species showed no uptake of cobalamin bound to transport proteins, although the latter type of cobalamin by far predominates in vivo. Certain free-living and parasitic helminths in vitro and in vivo took up a variety of folyl and antifolyl monoglutamates, but it is not known whether helminths can take up any folyl polyglutamates. Helminths that have any folate-dependent metabolism appear able to produce polyglutamylated forms of the required tetrahydrofolate coenzymes. Helminths that possess a functional cobalamin-dependent pathway from succinyl CoA to propionyl CoA appear able to form the required adenosylcobalamin coenzyme. Only free-living helminths may possess a cobalamin (and 5-methyltetrahydrofolate)-dependent pathway from homocysteine to methionine. It is likely that all helminths possess the 5,10-methylenetetrahydrofolate-dependent pathway from deoxyuridylate to thymidylate. All helminths appear able to salvage purine bases and nucleosides, but 10-formyltetrahydrofolate-dependent de novo purine ribonucleotide synthesis has been demonstrated unequivocally only in nematodes. The primitive parasitic groups of helminths exhibit cobalamin metabolism, whereas the more highly evolved ones seem to have lost the mechanisms for uptake and the associated biochemical pathways utilizing cobalamins.

Involvement of tetrahydrofolate cofactors in de novo purine ribonucleotide synthesis by adult Brugia pahangi and Dirofilaria immitis.

Adult Brugia pahangi in vitro, unlike mouse leukemia L1210 cells, converted 5-methyltetrahydrofolate (CH3FH4) directly to 5,10-methylenetetrahydrofolate and thence to other FH4 cofactors. The excreted CO2 that was derived from CH3FH4 was due to the presence within the filariae of 10-formyltetrahydrofolate dehydrogenase (EC 1.5.1.6) which catalyzes the deformylation of 10-formyl-tetrahydrofolate. Adult B. pahangi and Dirofilaria immitis, incubated in a purine-free medium containing [5-14C]CH3FH4 as the only form of folate, synthesized purine ribonucleotides radiolabeled at positions 2 and 8 of the purine ring. Presumably, 10-formyl[14C]FH4 donated Carbon 2 during the synthesis de novo of the purine ring and 5,10-methenyl[14C]FH4 donated Carbon 8.

Pathways of purine ribonucleotide biosynthesis in the adult worm Metastrongylus apri (Nematoda: Metastrongyloidea) from pig lung.

The pathways of purine ribonucleotide synthesis and interconversion that are operative in the intact adult pig lung worm Metastrongylus apri were identified by radioisotope tracing. The rate of [14C]glycine incorporation into purines was low but sufficient to demonstrate synthesis de novo. Radioactively labelled adenine, hypoxanthine and guanine were readily taken up and converted to the corresponding mononucleotides. Most of the AMP and GMP formed were phosphorylated to the triphosphates. These two nucleotides were interconvertable by pathways in which IMP is an intermediate. Adenosine was converted to nucleotides by direct phosphorylation as well as via formation of hypoxanthine. The rate of synthesis of adenine nucleotides from hypoxanthine was 5-7 times that of guanine nucleotides; conversion of IMP to AMP and to xanthosine 5'-monophosphate were the rate-limiting steps.

Purine ribonucleotide biosynthesis in gravid Angiostrongylus cantonensis (Nematoda: metastrongyloidea)

1. The pathways of purine ribonucleotide synthesis and interconversion that are operative in the gravid rat lung worm, Angiostrongylus cantonensis, were identified by radioisotope tracing in intact whole worms and by measurement of enzyme activities in cell-free extracts 2. Ribonucleotides were rapidly formed by utilization of adenine, hypoxanthine and guanine and by direct phosphorylation of adenosine. 3. AMP and GMP were interconvertible but these reactions were very slow. 4. The synthetic capacities of purine ribonucleotide metabolism were much higher than the catabolic functions in the parasite at this particular stage of the life cycle.

Effects of 5'-deoxyadenosine on phosphoribosyl pyrophosphate and purine nucleotide synthesis in ehrlich ascites tumor cells in vitro

Abstract 5'-Deoxyadenosine inhibits purine ribonucleotide synthesis de novo , as well as synthesis from adenine, guanine and hypoxanthine in Ehrlich ascites tumor cells in vitro . This is due to inhibition of the synthesis of phosphoribosyl pyrophosphate, but phosphoribosyl pyrophosphate synthetase itself is not inhibited. 5'-Deoxyadenosine affects several aspects of glucose metabolism, and it is proposed that this leads, in an as yet unknown way, to a decreased availability of ribose 5-phosphate for phosphoribosyl pyrophosphate synthesis.

Specificity of adenosine deaminase inhibitors

Abstract The specificity of the potent adenosine deaminase inhibitors deoxycoformycin (covidarabine), coformycin and erythro -9-(2-hydroxy-3-nonyl)adenine (EHNA), has been assessed in Ehrlich ascites tumor cells in vitro and in cultured mouse lymphoma L5178Y cells. EHNA is both less potent an inhibitor of adenosine deaminase than deoxycoformycin, and is less specific. High concentrations of deoxycoformycin and EHNA inhibit all pathways of purine ribonucleotide synthesis, and inhibit the conversion of inosinate to adenine and guanine nucleotides. These drugs also inhibit purified adenylate deaminase, but inhibition of this enzyme in intact cells can only be detected at high rates of deamination of adenylate. Deoxycoformycin potentiates the toxicity of adenine against cultured cells.

Purine metabolism in Saccharomyces cerevisiae

The synthesis, interconversion, and catabolism of purine bases, ribonucleosides, and ribonucleotides in wild-type Saccharomyces cerevisiae were studied by measuring the conversion of radioactive adenine, hypoxanthine, guanine, and glycine into acid-soluble purine bases, ribonucleosides, and ribonucleotides, and into nucleic acid adenine and guanine. The pathway(s) by which adenine is converted to inosinate is (are) uncertain. Guanine is extensively deaminated to xanthine. In addition, some guanine is converted to inosinate and adenine nucleotides. Inosinate formed either from hypoxanthine or de novo is readily converted to adenine and guanine nucleotides.

Effect of lowered intracellular ATP and GTP concentrations on purine ribonucleotide synthesis and interconversion

The effects of lowered intracellular ATP and GTP concentrations on enzymes of purine ribonucleotide synthesis and intercoversion were studied using intact Ehrlich ascites tumor cells. The apparent rates of phosphoribosyl pyrophosphate synthetase (EC 2.7.6.1) and of inosinate dehydrogenase (EC 1.2.1.14) were increased in cells containing lowered purine nucleotide concentrations, but apparent activities of amidophosphoribosyltransferase (EC 2.4.2.14), the purine phosphoribosyltransferases, and other enzymes of purine ribonucleotide interconversion were not affected.

Effects of actinomycin D on purine ribonucleotide metabolism in Ehrlich ascites tumor cells in vitro.

Actinomycin D treatment of Ehrlich ascites tumor cells in vitro causes slight to moderate inhibition of purine ribonucleotide synthesis de novo and from purine bases, and strong inhibition of inosinate dehydrogenase activity. These effects have the same dose–response relationship as inhibition of RNA synthesis by this drug. Daunomycin has similar effects on purine metabolism at a concentration that substantially inhibits nucleic acid synthesis. Actinomycin D treatment leads to elevated intracellular concentrations of ATP and GTP, and the effects of this drug on purine metabolism are believed to be mediated by these purine ribonucleoside triphosphates.

Mechanism of antiuric action of 4-oxy- and 4-thiopyrazolopyrimidines

Abstract Among the pyrazolopyrimidine derivatives used in hyperuricemia, thiopurinol [4-mercaptopyrazolo-(3,4-d) pyrimidine] exhibits different therapeutic effects than those of allopurinol [4-hydroxypyrazolo-(3,4-d) pyrimidine] and oxypurinol [4,6-dihydroxypyrazolo-(3,4-d) pyrimidine] which leads us to assume that its hypouricemic mechanism is different. The action of the three derivatives has been compared: (a) as xanthine oxydase inhibitors: thiopurinol activity is intermediate between that of the two others, (b) as subtrates of the phosphoribosyl transferase system: the nucleotide of thiopurinol has been isolated and identified, (c) as natural substrate HGPRT inhibitors (guanine and hypoxanthine). Finally, their action on PRPP consumption and on its intracellular level has been specified. In the absence of precise data on the concentrations realized in vivo with the therapeutic doses used, the interpretation of enzymatic kinetic data does not appear significant. It seems, however, that allopurinol and thiopurinol reduce de novo synthesis of purine ribonucleotides by depressing the PRPP intracellular level.

A kinetic analysis of purine nucleotide synthesis and interconversion in intact Ehrlich ascites tumor cells.

AbstractA pseudo‐first order kinetic analysis has described the reactions of purine ribonucleotide synthesis and interconversion in Ehrlich ascites tumor cells in vitro. Rate constants were determined for 13 reactions at non‐saturating precursor concentration (50 μM); nucleotide formation from adenine‐14C and hypoxanthine‐14C was rate limiting for ATP synthesis and nucleotide formation from guanine‐14C and hypoxanthine‐11C was rate limiting for GTP synthesis. The initial rites of nucleotide synthesis from 1.0 mM adenine‐14C and hypoxanthine‐14C were equivalent to the rates of nucleotide synthesis at non‐saturating base concentration, but the rate of nucleotide synthesis at saturating base concentration after 30 minutes incubation was 20‐25% that of non‐saturating precursor concentration.

Adenosine metabolism in Micrococcus sodonensis.

Enzymes required for the synthesis of purine ribonucleotides from exogenous adenosine, inosine, and guanosine were shown to be present constitutively in Micrococcus sodonensis. An indirect pathway for the synthesis of adenine nucleotides from adenosine was suggested by the high intracellular adenosine deaminase activity. Concentrations of intracellular adenine nucleotides were elevated in the presence of exogenous adenosine and under those conditions direct measurements of the activity of the pathway for de novo purine ribonucleotide biosynthesis indicated that the activity of that pathway was almost completely inhibited.

Effects of elevated intracellular ATP and GTP concentrations on purine ribonucleotide synthesis and interconversion.

The effects of elevated intracellular ATP and GTP concentrations on enzymes of purine ribonucleotide synthesis and interconversion were examined in intact Ehrlich ascites tumor cells. Elevated ATP and GTP concentrations inhibit nucleotide synthesis from purine bases up to 50% and elevated concentrations of GTP severely inhibit IMP dehydrogenase (60%). In contrast, adenylate deaminase activity was inhibited 16% at the highest concentration of GTP studied and elevated ATP concentrations had only a slight inhibitory effect on adenylosuccinate synthetase plus lyase (9%).

Regulation of purine ribonucleotide synthesis by end product inhibition. 3. Effect of purine nucleotides on succino-AMP synthetase of Bacillus subtilis.

Regulation of purine ribonucleotide synthesis by end product inhibition. 3. Effect of purine nucleotides on succino-AMP synthetase of Bacillus subtilis.

Regulation of purine ribonucleotide synthesis by end product inhibition. II. Effect of purine nucleotides on phosphoribosylpyrophosphate amidotransferase of Bacillus subtilis.

Regulation of purine ribonucleotide synthesis by end product inhibition. II. Effect of purine nucleotides on phosphoribosylpyrophosphate amidotransferase of Bacillus subtilis.

Inhibition of glycolysis in Ehrlich ascites tumor cells incubated with formimino-L-glutamate.

Incubation of tumor cells with formiminoglutamate leads to inhibition of lactate synthesis from glucose, inhibition of phosphoribosyl pyrophosphate synthesis, and inhibition of purine ribonucleotide synthesis de novo and from purine bases. Lactate synthesis from inosine was increased. These effects may result from inhibition of triose phosphate isomerase by a metabolite of formiminoglutamate.

Regulation of purine ribonucleotide synthesis by end product inhibition. I. Effect of purine nucleotides on inosine-5'-phosphate dehydrogenase, xanthosine-5'-phosphate aminase and adenylosuccinate lyase of Bacillus subtilis.

Regulation of purine ribonucleotide synthesis by end product inhibition. I. Effect of purine nucleotides on inosine-5'-phosphate dehydrogenase, xanthosine-5'-phosphate aminase and adenylosuccinate lyase of Bacillus subtilis.