Purine metabolism in Trypanosoma cruzi

Culture forms of Trypanosoma cruzi are incapable of synthesizing purines de novo from formate, glycine, or serine and require an exogenous purine for growth. Adenine, hypoxanthine, guanine, xanthine and their respective ribonucleosides are equal in their abilities to support growth. Radiolabeled purine bases, with the exception of guanine, are stable and are converted to their respective ribonucleotides directly by phosphoribosyltransferase activity. Guanine is both converted to its ribonucleotide and deaminated to xanthine. Purine nucleosides are not hydrolysed to any extent but are converted to their respective ribonucleotides. This conversion may involve a rate-limiting ribonucleoside cleaving activity or a purine nucleoside kinase or phosphotransferase activity. The apparent order of salvage efficiency for the bases and their respective ribonucleosides is adenine > hypoxanthine > guanine > xanthine.     

Mode of action of 2-amino-6-chloro-1-deazapurine

2-Amino-6-chloro-1-deazapurine is of interest as a purine analog with demonstrated in vivo activity against mouse leukemia L1210. That the active form of this agent is a nucleotide and that the nucleotide is formed by the action of hypoxanthine (guanine) phosphoribosyltransferase were shown by the facts that (a) L1210 cells deficient in hypoxanthine phosphoribosyltransferase were insensitive to the analog; (b) hypoxanthine, but not adenine, prevented the formation of the analog nucleotide by enzyme preparations containing activities of both hypoxanthine and adenine phosphoribosyltransferases; and (c) the cytotoxicity of the analog was prevented by hypoxanthine. The ribonucleoside of this analog was not toxic to cell cultures and hence is not phosphorylated or cleaved to the base. In intact HEp-2 cells and L1210 cells, the analog was metabolized to the nucleoside 5'-phosphate which accumulated to concentrations as high as 1000 nmoles/10(9) cells; no di- or triphosphates were detected. In HEp-2 cells, the analog reduced the pools of purine nucleotides with some accumulation of IMP. The toxicity of minimal inhibitory concentrations of the analog to HEp-2 cells could be prevented or reversed by 4(5)-amino-5(4)-imidazolecarboxamide (AIC); the toxicity of higher concentrations could be prevented or reversed by a combination of adenine and guanosine but not by AIC. The analog inhibited the incorporation of formate into purine nucleotides and into macromolecules at concentrations that had no effect on utilization of hypoxanthine; at higher concentrations the incorporation of hypoxanthine was inhibited. Low concentrations also inhibited the utilization of uridine and thymidine. The incorporation of hypoxanthine and AIC into guanine nucleotides, but not adenine nucleotides, was inhibited. These results indicate two sites of inhibition of the biosynthesis of purine nucleotides, the more sensitive one being on an early step of the pathway and the less sensitive one on the IMP-GMP conversion. That the blockade of de novo synthesis probably was at the site of feedback inhibition was indicated by the fact that the analog inhibited the accumulation of formylglycinamide ribonucleotide in azaserine-treated cells but did not inhibit the synthesis of 5'-phosphoribosyl 1-pyrophosphate. Comparative studies were performed with the related analog, 2-amino-6-chloropurine, which has been reported to produce a similar dual blockade of the purine pathway. This purine was less toxic than its 1-deaza analog; it produced a modest decrease in adenine nucleotides but increased pools of guanine nucleotides.(ABSTRACT TRUNCATED AT 400 WORDS)     

Nucleosides of 2-aza-purines— cytotoxicities and activities as substrates for enzymes metabolizing purine nucleosides

Nucleosides of 2-aza-adenine, 2-aza-hypoxanthine and 4-amino-7H-pyrazolo[3,4-d]-v-triazine were examined: (a) for cytotoxicity to cultured H. Ep. No. 2 cells and to sublines resistant to certain analogs of purines and purine nucleotides and deficient in certain enzymes of purine metabolism, and (b) as substrates for adenosine kinase, adenosine deaminase, and nucleoside-cleaving enzymes. 2-Aza-adenosine was much more toxic than 2-aza-adenine and was a good substrate for both the kinase and the deaminase. The responses of the resistant cell lines indicated that the cytotoxicity of 2-aza-adenosine was due both to its direct phosphorylation and to its conversion to 2-aza-hypoxanthine. 2-Aza-inosine, 2′-deoxy-2-aza-inosine and 2-aza-hypoxanthine had similar cytotoxicities, and the responses of the resistant cell lines showed that the cytotoxicities of both of the nucleosides resulted from their conversion to 2-aza-hypoxanthine. The ribonucleoside of 4-amino-7H-pyrazolo[3,4-d]-v-triazine was less toxic than 2-aza-adenosine, and was a good substrate for adenosine kinase but a poor substrate for adenosine deaminase. 2-Aza-inosine and 2′-deoxy-2-aza-inosine were cleaved to 2-aza-hypoxanthine by cell-free supernatants, but the conversions were poor relative to those of inosine and 2′-deoxyinosine. 3′-Deoxy-2-aza-adenosine and the α-arabinosyl, β-arabinosyl and β-xylosyl derivatives of 2-aza-adenine were not toxic. 3′-Deoxy-2-aza-adenosine and β-xylosyl-2-aza-adenine were moderately good substrates for the deaminase, but α-arabinosyl-2-aza-adenine was not deaminated and β-arabinosyl-2-aza-adenine was deaminated at a rate less than 5 per cent that of β-arabinosyladenine. These results indicate that some of these nucleosides, particularly 2-aza-adenosine, may merit further study as growth-inhibitory and potential antitumor agents.     

Inosine analogs as substrates for adenosine kinase--influence of ionization of the N-1 proton on the rate of phosphorylation.

This study was undertaken to attempt to rationalize previously obtained and apparently conflicting findings that although adenosine kinase (EC 2.7.1.20) from H.Ep. # 2 cells did not accept inosine as a substrate, these cells became resistant to an inosine analog, 8-azainosine, only when activities of both hypoxanthine (guanine) phosphoribosyltransferase [H(G)PRTase] and adenosine kinase were lost. No evidence could be found for the presence of inosine kinase in H.Ep. # 2 cells: crude supernatants from these cells converted ring-labeled inosine to IMP, but the conversion was prevented by the addition of hypoxanthine and therefore apparently was achieved by the alternative pathway involving the action of H(G)PRTase on hypoxanthine. An investigation of inosine and inosine analogs as substrates for adenosine kinase revealed that certain inosine analogs were substrates and that the substrate activity could be correlated with the degree of ionization of the N-1 proton. Of four 6-oxo and 6-thio nucleosides studied. 8-aza-6-thioinosine had the lowest pK_a (6.75) and was the only one that was a good substrate at pH 7.0; the K_m was 210μM and the V_max was about 1.5 times that of adenosine. The rate of phosphorylation of 8-aza-6-thioinosine increased markedly as the pH of the reaction mixture was increased in the pH range 6.0 to 7.0. Phosphorylation of 8-azainosine (pK_a 7.45) and 6-thioinosine (pK_a 7.60) was much poorer and could be demonstrated at pH 7.0 only after overnight incubation with the kinase. The fact that 8-azainosine and 8-aza-6-thioinosine are substrates whereas ionsine is not, can be rationalized by the facts that (a) the substitution of an N-atom for the C-8 atom of the nucleoside lowers the pK_a so that the N-1 proton is more strongly ionized at physiological pH; and (b) the ionized form of a 6-oxo or 6-thio nucleoside resembles adenosine with respect to the bond structure at the 1- and 6-positions of the purine ring whereas the unionized form does not.     

Purine metabolism in Leishmania donovani amastigotes and promastigotes

Purine metabolism in Leishmania donovani amastigotes was found to be similar to that of promastigotes with the exception of adenosine metabolism. Adenosine kinase activity in amastigotes is approximately 50-fold greater than in promastigotes. Amastigotes deaminate adenosine to inosine through adenosine deaminase, an enzyme not present in promastigotes. Inosine is cleaved to hypoxanthine and phosphoribosylated by hypoxanthine-guanine phosphoribosyltransferase. Promastigotes cleave adenosine to adenine and deaminate adenine to hypoxanthine via adenase, an enzyme not present in amastigotes. Hypoxanthine is phosphoribosylated by hypoxanthine-guanine phosphoribosyltransferase.     

Inosine 5′-monophosphate dehydrogenase from Sarcoma 180 cells—Substrate and inhibitor specificity

The substrate and inhibitor specificity of IMP dehydrogenase from Sarcoma 180 ascites tumor cells has been studied with twenty purine nucleotide analogs. Several were found to be substrates with the following efficiencies (V_max/K_m):IMP (4000), 8-azaIMP (1360), 6-thioIMP (250), araIMP (250) and dIMP (240). While substrate activity was not detected with the 5′-phosphates of 6-methylmercaptopurine riboside or 1-ribosylallopurinol (rates less than 1/10,000th that of IMP), they were competitive inhibitors with respect to IMP (both with K_i, values of 0.43 mM). Seven XMP analogs and six GMP analogs were also found to be competitive inhibitors with respect to IMP. Methods for the synthesis of the 5′-phosphates of arabinosylhypoxanthine. arabinosylxanthine. arabinosylguanine and 2′-deoxyxanthosine are described.     

Enzymes of purine and pyrimidine metabolism from the human malaria parasite, Plasmodium falciparum

Plasmodium falciparum trophozoites were isolated by mechanical rupture of infected human erythrocytes followed by a series of differential centrifugation steps. After lysis with sonication, the 100 000 x g supernatant of parasites and uninfected host cells was used to determine the specific activities of a number of enzymes involved in purine and pyrimidine metabolism. P. falciparum possessed the purine salvage enzymes: adenosine deaminase, purine nucleoside phosphorylase, hypoxanthine-guanine phosphoribosyltransferase (PRTase), xanthine PRTase, adenine PRTase, adenosine kinase. The last two enzymes, however, were present at much lower activity levels. Hypoxanthine was converted (presumably via IMP) into adenine and guanine nucleotides only in the presence both of supernatant and membrane fractions of P. falciparum. Two enzymes involved in the de novo synthesis of pyrimidines, orotic acid PRTase, and orotidine 5'-phosphate decarboxylase, were present in parasite extracts as were the enzymes for pyrimidine nucleotide phosphorylation: UMP-CMP kinase, dTMP kinase, nucleoside diphosphate kinase. Xanthine oxidase, CTP synthetase, cytidine deaminase and several kinases for the salvage of pyrimidine nucleosides were not detected in the parasites. Both phosphoribosyl pyrophosphate synthetase and uracil PRTase were present but at low activity levels. Human erythrocytes displayed similar but not identical enzyme patterns. Enzyme specific activities, however, were generally much lower than those of the corresponding parasite enzymes.