RESUMO
This paper describes for the first time the isolation and characterization of a high-molecular-weight predominant DNA polymerase from the genus Leishmania, which are parasitic flagellated protozoa. Like mammalian DNA polymerase alpha, the leishmanial DNA polymerase, designated DNA polymerase A, is of high-molecular-weight, is sensitive to N-ethylmaleimide and is inhibited by high ionic strength. Unlike mammalian DNA polymerase alpha, but similar to the predominant DNA polymerase isolated from the related lower eukaryotic organisms, Trypanosoma cruzi and Crithidia fasciculata, the leishmanial DNA polymerase A is resistant to inhibition by aphidicolin, a potent inhibitor of DNA replication in mammalian cells and of DNA polymerase alpha. The DNA polymerase A was purified 28,000-fold and properties such as pH optimum, salt sensitivity, template requirements and response to DNA polymerase inhibitors were determined. A low-molecular-weight DNA polymerase was detected during the isolation procedures, but was separated from the polymerase A activity. Differences in responses to specific antisera and specific mammalian DNA polymerase alpha inhibitors suggest that the leishmanial high-molecular-weight A enzyme is sufficiently different to suggest this enzyme as a chemotherapeutic target.
Assuntos
DNA Polimerase Dirigida por DNA/isolamento & purificação , Leishmania mexicana/enzimologia , Proteínas de Protozoários/isolamento & purificação , Animais , Afidicolina/farmacologia , DNA Polimerase Dirigida por DNA/química , Estabilidade Enzimática , Temperatura Alta , Humanos , Focalização Isoelétrica , Leishmania mexicana/genética , Peso Molecular , Inibidores da Síntese de Ácido Nucleico , Concentração Osmolar , Proteínas de Protozoários/antagonistas & inibidores , Proteínas de Protozoários/química , Moldes GenéticosRESUMO
4-Amino-5-imidazolecarboxamide, but not its riboside or ribotide, is inhibitory to the growth of promastigotes of Leishmania donovani, L. braziliensis, L. tarentolae, and L. mexicana, eventually causing cell lysis. Conversely, it is not inhibitory to the growth of epimastigotes of Trypanosoma cruzi. This substituted imidazole proved to be an excellent inhibitor of guanine deaminase from all of the trypanosomatids used in this study, with Ki values in the microM range.
Assuntos
Aminoimidazol Carboxamida/farmacologia , Imidazóis/farmacologia , Leishmania/efeitos dos fármacos , Trypanosoma cruzi/efeitos dos fármacos , Aminoimidazol Carboxamida/análogos & derivados , Animais , Guanina Desaminase/antagonistas & inibidores , Leishmania/enzimologia , Leishmania/crescimento & desenvolvimento , Ribonucleosídeos/farmacologia , Trypanosoma cruzi/enzimologia , Trypanosoma cruzi/crescimento & desenvolvimentoRESUMO
RNA, DNA and protein biosynthesis were studied in Leishmania donovani and L. tropica promastigotes cultured with or without sinefungin. Thymidine incorporation was significantly impaired by this compound. Neither the uptake of thymidine nor its phosphorylation were inhibited. Furthermore the ratios of deoxyribonucleotide to the corresponding ribonucleotide were not significantly affected by sinefungin. Analysis of the DNA indicates that the inhibition of thymidine incorporation affects mostly nuclear DNA, kDNA being less affected by this drug. No such effect on thymidine incorporation was observed in macrophages, the host cells of these parasites.
Assuntos
Adenosina/análogos & derivados , Replicação do DNA/efeitos dos fármacos , Leishmania/efeitos dos fármacos , Adenosina/farmacologia , Animais , Linhagem Celular , Leishmania/genética , Macrófagos/metabolismo , Camundongos , Fosforilação , Biossíntese de Proteínas/efeitos dos fármacos , S-Adenosil-Homocisteína/farmacologia , S-Adenosilmetionina/farmacologia , Especificidade da Espécie , Timidina/metabolismo , Transcrição Gênica/efeitos dos fármacosRESUMO
Two previously isolated DNA polymerases from the parasitic protozoan Leishmania mexicana were further characterized by exposure to inhibitors of mammalian DNA polymerases. DNA polymerase A, a high molecular mass enzyme, and DNA polymerase B, a beta-like DNA polymerase were compared to each other and to their mammalian counterparts regarding pH optimum, utilization of templates, and response to various inhibitors and ionic strengths. The results suggest that the DNA polymerases from L. mexicana differ from the host enzymes and may offer a target for chemotherapeutic intervention.
Assuntos
DNA Polimerase III/metabolismo , DNA Polimerase I/metabolismo , Leishmania mexicana/enzimologia , Animais , DNA Polimerase I/antagonistas & inibidores , DNA Polimerase I/isolamento & purificação , DNA Polimerase III/antagonistas & inibidores , DNA Polimerase III/isolamento & purificação , Concentração de Íons de Hidrogênio , Peso Molecular , Cloreto de Potássio/farmacologia , Cloreto de Sódio/farmacologia , Moldes GenéticosRESUMO
The purine phosphoribosyltransferases of Crithidia fasciculata were identified and some of their properties described. The organism possesses three separate enzymes for the production of AMP, IMP, and GMP. The evidence for this comes from the observed differences in elution patterns from gel filtration columns, differences in heat sensitivity, and especially the clear separation of hypoxanthine phosphoribosyltransferase from guanine phosphoribosyltransferase by affinity chromatography on GMP-agarose. APRTase is activated most efficiently by Zn++, whereas HPRTase and GPRTase are activated most effectively by Co++. In no case did the product mononucleotides produce strong inhibition of the transferase activities.
Assuntos
Adenina Fosforribosiltransferase/metabolismo , Crithidia/enzimologia , Hipoxantina Fosforribosiltransferase/metabolismo , Pentosiltransferases/metabolismo , Animais , Sistema Livre de Células , Cobalto/farmacologia , Ativação Enzimática , Temperatura Alta , Magnésio/farmacologia , Manganês/farmacologia , Especificidade por Substrato , Zinco/farmacologiaRESUMO
Sinefungin, a natural nucleoside isolated from cultures of Streptomyces incarnatus and S. griseolus, is structurally related to S-adenosylhomocysteine and S-adenosylmethionine. Sinefungin has been shown to inhibit the development of various fungi and viruses, but its major attraction to date resides in its potent antiparasitic activity. This compound has been reported to display antiparasitic activity against malarial, trypanosomal, and leishmanial species. Very little is known about the antiparasitic mode of action of sinefungin. We found that S-adenosylmethionine was capable of reversing the inhibitory growth effects of sinefungin in Leishmania mexicana and that dATP was capable of reversing inhibitory effects of the drug on DNA polymerase activity when pyrophosphate release was measured. However, when incorporation of [3H]dTTP was used to measure DNA polymerase activity, no inhibition could be observed. Inhibition of DNA polymerase activity by sinefungin occurred only during the initial stages of purification of this enzyme, and inhibition by aphidicolin, a known DNA polymerase inhibitor, paralleled the inhibition by sinefungin. Neither sinefungin nor aphidicolin inhibited partially purified DNA polymerase. S-Adenosylmethionine synthetase was partially purified, and sinefungin, at levels active in vitro, had no significant effect. Sinefungin was significantly suppressive against both L. donovani and L. braziliensis panamensis infections in hamsters when compared with meglumine antimonate (Glucantime).
Assuntos
Adenosina/análogos & derivados , Antimaláricos/farmacologia , Leishmania mexicana/efeitos dos fármacos , Adenosina/farmacologia , Adenosina/uso terapêutico , Adenosina/toxicidade , Animais , Antimaláricos/uso terapêutico , Antimaláricos/toxicidade , Afidicolina , Células Cultivadas , Cricetinae , DNA Polimerase II/antagonistas & inibidores , Diterpenos/farmacologia , Leishmania mexicana/enzimologia , Masculino , Mesocricetus , Testes de Mutagenicidade , Inibidores da Síntese de Ácido Nucleico , Biossíntese de ProteínasRESUMO
N6-methyladenine (6-methylaminopurine [6-MA]), a plant growth regulator and a normal constituent of nucleic acids, has been found to inhibit the growth of Trypanosoma cruzi, Leishmania braziliensis, L. donovani, L. tarentolae, L. mexicana, and Crithidia fasciculata. The extent of growth inhibition in these organisms is related to the sensitivity of guanine deaminase (guanine aminohydrolase, EC 3.5.4.3), adenine deaminase (adenine aminohydrolase, EC 3.5.4.2), and adenosine hydrolase and phosphorylase. 6-MA was not an inhibitor of the purine phosphoribosyltransferases. Of the trypanosomid flagellates tested. Trypanosoma cruzi was most susceptible to 6-MA. Neither adenine deaminase (as found in the leishmaniae and C. fasciculata) nor adenosine deaminase (as found in mammalian cells) could be demonstrated in T. cruzi. Guanine deaminase, which is strikingly inhibited by 6-MA in T. cruzi, appears to play a major role in the purine salvage pathway of this organism, as judged from growth experiments and enzyme inhibition studies. Enzyme sensitivities to 6-MA vary greatly depending upon the organism. Rabbit liver guanine deaminase was shown to be insensitive to 6-MA at the concentrations used in this study.
Assuntos
Adenina/análogos & derivados , Aminoidrolases/antagonistas & inibidores , Antiprotozoários/farmacologia , Eucariotos/efeitos dos fármacos , Guanina Desaminase/antagonistas & inibidores , Adenina/metabolismo , Adenina/farmacologia , Inibidores de Adenosina Desaminase , Animais , Crithidia/efeitos dos fármacos , Remoção de Radical Alquila , Eucariotos/enzimologia , Eucariotos/crescimento & desenvolvimento , Leishmania/efeitos dos fármacos , Fígado/enzimologia , Purinas/metabolismo , Coelhos , Trypanosoma cruzi/efeitos dos fármacosRESUMO
Deoxyribonucleic acid polymerase beta (EC 2.7.7.7) from the lower eukaryotic parasitic protozoan Leishmania mexicana has been partially purified over 9,000 fold and characterized for the very first time. Like mammalian DNA polymerase beta the protozoan enzyme is of low molecular weight (40,000), has a broad pH range, and is resistant to inhibition by N-ethylmaleimide and aphidicolin. It is unlike mammalian DNA polymerase beta in utilization of various templates and response to various inhibitors and sensitivity to high ionic strength, but similar to a beta-like enzyme from a related organism Crithidia fasciculata. It is estimated that this enzyme constitutes 20% of the polymerase activity of the crude cell extract.
Assuntos
DNA Polimerase I/isolamento & purificação , Leishmania mexicana/enzimologia , Animais , Centrifugação com Gradiente de Concentração , Cromatografia em Gel , Reações Cruzadas/imunologia , DNA Polimerase I/efeitos dos fármacos , Mamíferos , Peso MolecularRESUMO
Eleven medications used in the treatment of the human immunodeficiency virus or subsequent opportunistic infections were tested to determine their toxicity to the growth of the CEM line of transformed CD4+ T-cells. A selectivity ratio (IC50/EC50) for each agent against its target pathogen was calculated as an in vitro indication of the therapeutic value of that agent. Among the anti-HIV agents tested in this study, 2',3'-dideoxyinosine (ddI) had the highest in vitro selectivity ratio, 2.5 times higher than that of 3'-azido-3'-deoxythymidine (AZT). Dapsone had the highest selectivity ratio of the four agents used in the treatment of Pneumocystis carinii pneumonia that were tested. Ketoconazole had a very low selectivity ratio for Candida spp. due to its very high toxicity to CD4+T-cells.
Assuntos
Síndrome da Imunodeficiência Adquirida/tratamento farmacológico , Antivirais/toxicidade , Linfócitos T CD4-Positivos/efeitos dos fármacos , Didanosina/toxicidade , Humanos , Leucemia , Células Tumorais Cultivadas/efeitos dos fármacos , Zidovudina/toxicidadeRESUMO
A DNA-dependent RNA polymerase has been isolated and characterized from the parasitic flagellated protozoan Leishmania mexicana. The initial stages of purification utilized high-ionic-strength extraction and protamine sulfate treatment. The enzyme was further purified by differential elution by heparin-Sepharose, DEAE-Sephadex, and carboxymethyl-Sephadex chromatography. Analysis of the chromatographically purified RNA polymerase on nondenaturing gels revealed two electrophoretic forms. The enzyme isolated had characteristics of true DNA-dependent RNA polymerase since it required DNA and all four nucleoside triphosphates for synthesis of RNase-sensitive products. Analysis of ammonium sulfate and metal ion optima, as well as relative activities of the enzyme with Mn2+ versus Mg2+, gave results similar to those reported for other RNA polymerase IIIs in eucaryotes. Formycin A triphosphate was found to be a noncompetitive inhibitor of RNA polymerase III, and cordycepin triphosphate was found to be inhibitory, although the exact mode of inhibition was not determined.
Assuntos
Antibióticos Antineoplásicos/farmacologia , RNA Polimerases Dirigidas por DNA/isolamento & purificação , Nucleotídeos de Desoxiadenina/farmacologia , Formicinas/farmacologia , Leishmania mexicana/enzimologia , RNA Polimerase III/isolamento & purificação , Ribonucleotídeos/farmacologia , Animais , Cromatografia , RNA Polimerase III/antagonistas & inibidoresRESUMO
Caffeine (2 mg/mL) inhibited the incorporation of [14C]adenine into actively growing cells of Clostridium perfringens NCTC 8679 in a dose-dependent manner. Also reduced by caffeine was incorporation of [14C]thymidine and 14C-labeled amino acids. No effect on guanine, uracil, adenosine, guanosine, or uridine was detected. Actual incorporation of [14C]caffeine or [14C]thymine in control cultures did not occur.
Assuntos
Proteínas de Bactérias/biossíntese , Cafeína/farmacologia , Clostridium perfringens/efeitos dos fármacos , DNA Bacteriano/biossíntese , RNA Bacteriano/biossíntese , Adenina/metabolismo , Aminoácidos/metabolismo , Clostridium perfringens/metabolismo , Clostridium perfringens/fisiologia , Enterotoxinas/biossíntese , Esporos Bacterianos , Timidina/metabolismoRESUMO
Xanthine phosphoribosyltransferase (XPRTase; EC 2.4.4.22) was found in the promastigotes of four species of Leishmania (L. mexicana, L. donovani, L. braziliensis and L. tarentolae). In no case was there any transribosylation from 5-phosphoribosyl-1-pyrophosphate (PRibPP), forming XMP, in dialyzed preparations, unless activated by a divalent cation. Magnesium and zinc were very low in activation efficiency in all cases, while manganese was optimally efficient. Cobalt was essentially equal to manganese for activation of the enzyme from L. mexicana and L. braziliensis but much less efficient for the enzyme from L. donovani and L. tarentolae. Gel filtration profiles of cell extracts of L. mexicana on Sephadex G-200 indicated that the enzymes catalyzing the transribosylation from PRibPP to guanine, hypoxanthine, and xanthine were inseparable. All were eluted near the void volume. The enzyme for adenine transribosylation was clearly separate. When cell extracts of L. mexicana were applied to Sephadex G-100 columns, the activity toward XMP formation from xanthine eluted with the void volume, together with a portion of that for the formation of GMP and IMP from guanine and hypoxanthine. A second peak of HGPRTase (EC 2.4.2.8) eluted somewhat later and was devoid of XPRTase activity. XPRTase from promastigotes of L. mexicana is heat labile, has rather a broad pH optima, and is stable to freezing when protected by nonspecific cell protein (40,000 g supernate as opposed to 100,000 g supernates).
Assuntos
Leishmania/enzimologia , Manganês/farmacologia , Pentosiltransferases/metabolismo , Animais , Cobalto/farmacologia , Ativação Enzimática , Cinética , Magnésio/farmacologia , Pentosiltransferases/isolamento & purificação , Temperatura , Xantinas/metabolismoRESUMO
In the presence of 100 micrograms of caffeine per ml or 200 micrograms of theobromine per ml, sporulation of Clostridium perfringens NCTC 8679 rose from less than 1 to 80 or 85%. Enterotoxin concentration increased from undetectable levels to 450 micrograms/mg of cell extract protein. Heat-resistant spore levels increased from less than 1,000 to between 1 X 10(7) and 2 X 10(7)/ml. These effects were partially reversible by the addition of adenosine or thymidine. In the case of NCTC 8238, caffeine and theobromine caused a three- to fourfold increase in the percentages of cells possessing refractile spores and a similar increase in enterotoxin concentration. Heat-resistant spore levels, however, were unaffected. Inosine was ineffective in promoting sporulation in NCTC 8679.
Assuntos
Cafeína/farmacologia , Clostridium perfringens/metabolismo , Enterotoxinas/biossíntese , Teobromina/farmacologia , Clostridium perfringens/efeitos dos fármacos , Relação Dose-Resposta a Droga , Nucleosídeos/farmacologia , Purinas/farmacologia , Pirimidinas/farmacologia , Esporos Bacterianos/efeitos dos fármacosRESUMO
Adenine aminohydrolase (EC 3.5.4.2) from four species of Leishmania and from Crithidia fasciculata was examined for specific activities, affinity for substrate (adenine), and stability to heat. All were found to be strongly and non-competitively inhibited by both coformycin and deoxycoformycin, two tight-binding inhibitors of adenosine deaminase (adenosine aminohydrolase, EC 3.5.4.4). Deoxycoformycin is the more potent inhibitor of the two. Neither inhibitor was active against the purine phosphoribosyltransferases. When deoxycoformycin was added to the defined growth medium containing hypoxanthine as the purine source, the growth of C. fasciculata was unaffected, but when adenine was the purine source for the organism, severe inhibition resulted. This implies that hypoxanthine is the obligatory base for nucleotide synthesis and that the adenine phosphoribosyltransferase (AMP:pyrophosphate phosphoribosyltransferase, EC 2.4.2.7) is, in some manner,idenied access to exogenous substrate.
Assuntos
Adenosina Desaminase/metabolismo , Crithidia/enzimologia , Leishmania/enzimologia , Nucleosídeo Desaminases/metabolismo , Inibidores de Adenosina Desaminase , Sítios de Ligação , Coformicina/análogos & derivados , Coformicina/farmacologia , Desoxirribonucleotídeos , Temperatura Alta , Desnaturação ProteicaRESUMO
A study of the enzymes of the orotate biosynthetic pathway in the kinetoplasid flagellate Crithidia fasciculata has revealed a number of differences between them and those of other organisms, either prokaryotic or eukaryotic. Carbamyl phosphate synthesis could not be demonstrated in cell-free extracts. However, the incorporation of both CO2 and the ureide carbon of citrulline into pyrimidines occurs in growing cells, the latter predominating over the former. The aspartate transcarbamylase of the flagellate has properties which are similar to those of this enzyme as it occurs in mammals rather than other microorganisms. Two enzymes, dihydroorotate synthetase and dihydroorotate hydrolase, are present, the former being responsible for the conversion of carbamylasparate to dihydroorotate. Dihydroorotate hydroxylase, a soluble enzyme requiring a reduced pteridine as a cofactor, converts dihydroorotate to orotate. The hydroxylase is inhibited by orotate, but not by pyrimidine or purine ribonucleotides. Thus orotate serves to control its own biosynthesis.
Assuntos
Eucariotos/enzimologia , Ácido Orótico/biossíntese , Amidoidrolases/metabolismo , Animais , Aspartato Carbamoiltransferase/metabolismo , Carbamoil-Fosfato Sintase (Amônia)/metabolismo , CinéticaRESUMO
The capacity for the rapid transport of purine bases by Crithidia fasciulata is found only in cells starved for purines. Cells grown in complete medium transport poorly. Rapid transport capability appears and then disappears during growth of purine-depleted cultures. This rapid transport appears to occur by a process of mediated diffusion. Two mechanisms are involved, one of low velocity and high affinity, the other of high velocity and low affinity. Accumulation of the bases within the cell occurs by their rapid conversion to ribonucleotides by phosphoribosyltransferases.
Assuntos
Adenina/metabolismo , Eucariotos/metabolismo , Guanina/metabolismo , Hipoxantinas/metabolismo , Nucleotídeos de Adenina/metabolismo , Animais , Azidas/farmacologia , Transporte Biológico , Cianetos/farmacologia , Dinitrofenóis/farmacologia , Nucleotídeos de Guanina/metabolismo , CinéticaRESUMO
Formycin B is a structural analog of inosine that is a potent inhibitor of Leishmania multiplication. Formycin B is reportedly converted to formycin A nucleotides and incorporated into RNA by the organisms, and it is unclear whether the active form of the drug is the nucleoside itself or its several metabolites. We confirmed that formycin A nucleotides are formed by formycin B-exposed L. mexicana promastigotes, and determined that the intraparasite concentration of Formycin B and its metabolites was 6 times the extracellular formycin B concentration. Formycin B did not significantly inhibit purine nucleoside transport by intact promastigotes or purine base phosphoribosylation by parasite lysates. Thus, the nucleoside does not appear to inhibit these initial steps of purine nucleoside metabolism. Since RNA and protein synthesis in formycin B-treated intact promastigotes was found to be inhibited within 30 minutes, the effect of formycin A metabolites on leishmanial protein synthesis was investigated in in vitro protein synthesis experiments. Messenger RNA from formycin B-treated promastigotes was translated only 40% as efficiently as control promastigote mRNA by rabbit reticulocyte lysates. In addition, when formycin A-5'-triphosphate was preincubated with the rabbit reticulocyte lysates, translation of control mRNA was 86% inhibited. Formycin B toxicity to Leishmania promastigotes appears to be at least partially due to inhibition of protein synthesis by formycin A nucleotides and formycin A containing mRNA.