J.Joseph Marr

Purine metabolism in Leishmania donovani and Leishmania braziliensis.

We have studied purine metabolism in the culture forms of Leishmania donovani and Leishmania braziliensis. These organisms are incapable of synthesizing purines de novo from glycine, serine, or formate and require an exogenous purine for growth. This requirement is better satisfied by adenosine or hypoxanthine than by guanosine. Both adenine and inosine are converted to a common intermediate, hypoxanthine, before transformation to nucleotides. This is due to the activity of an adenine aminohydrolase ((EC 3.5.4.2), a rather unusual finding in a eukaryotic cell. There is a preferential synthesis of adenine nucleotides, even when guanine or xanthine are used as precursors. The pathways of purine nucleotide interconversions in these Leishmania resemble those found in mammalian cells except for the absence of de novo purine biosynthesis and the presence of an adenine-deaminating activiting.

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 rete-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 greater than hypoxanthine greater than guanine greater than xanthine.

Purine and pyrimidine salvage pathways in Leishmania donovani

Leishmania donovani, grown in culture, salvaged radiolabeled purine bases which were distributed into adenine and guanine ribonucleotides and into the RNA of these cells. De novo synthesis of purines in L. donovani does not occur [J. J. Marr, R. L. Berens and D. J. Nelson, Biochim. biophys. Acta 544, 360 (1978)]. [8-14C]Adenine was rapidly deaminated to hypoxanthine via the action of an adenine aminohydrolase (EC 3.5.4.2). [8-14C]Guanine was also rapidly deaminated by guanase (EC 3.5.4.3) to form zanthine in these cells. Therefore, the formation of nucleotides of hypoxanthine and xanthine are the first committed steps of purine salvage in L. donovani. While purines are efficiently conserved by this parasite, the salvage of pyrimidines is not so dramatic. [2-14C]Orotic acid was converted to OMP and then incorporated into the pyrimidine nucleotides and into RNA, indicating the existence of the later steps of de novo pyrimidine synthesis. [6-14C]Thymidine was salvaged by L. donovani, being incorporated into the thymine deoxyribonucleotides and into DNA. The major pathway of thymidine metabolism in this parasite, however, was cleavage of the deoxyriboside linkage to form thymine, probably via the action of a thymidine phosphorylase (EC 2.4.2.4).

Purine metabolism in the bloodstream forms of trypanosoma gambiense and Trypanosoma rhodesiense

Bloodstream forms of Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense are incapable of de novo purine synthesis. Purine bases are converted directly to ribonucleotides and with the exception of guanine, are stable. Guanine is incorporated directly into ribonucleotides and also deaminated to xanthine. Purine ribonucleosides are hydrolyzed rapidly; these reactions may limit their incorporation since purine bases label the nucleotide pools more efficiently than do ribonucleosides. The apparent order of salvage efficiency for ribonucleosides is adenosine greater than inosine greater than guanosine greater than xanthosine for both organisms. T. b. gambiense salvages purine bases in the same order, while T. b. rhodesiense salvages purine bases in the order hypoxanthine greater than adenine greater than guanine greater than xanthine.

Purine metabolism in Trypanosoma brucei gambiense

Abstract The procyclic forms of Trypanosoma brucei gambiense do not incorporate glycine or serine into ribonucleotides. Although de novo purine synthesis does not occur, all purine bases and ribonucleotides are interconverted, indicating the presence of active salvage pathways. Guanine is actively deaminated to xanthine by guanase activity. Purine ribonucleosides are cleaved to their respective free bases. The order of salvage efficiency for purine bases and their respective ribonucleotides is: adenine > hypoxanthine > guanine > xanthine.

Leishmania donovani and Leishmania braziliensis: Hexokinase, glucose 6-phosphate dehydrogenase, and pentose phosphate shunt activity

Abstract The hexokinase and glucose 6-phosphate dehydrogenase of Leishmania donovani and L. braziliensis do not appear to be subject to metabolic regulation. The relative importance of glycolysis vs the pentose phosphate shunt is not clear, but experiments using specifically radiolabeled glucose and growth studies with carbohydrate-free media supplemented with ribose indicate that the pentose phosphate pathway is active in vivo.

Pyrazolopyrimidine metabolism in African trypanosomes: metabolic similarities to Trypanosoma cruzi and Leishmania spp.

Growth inhibition and radioisotope incorporation studies with allopurinol (4-hydroxypyrazolo(3,4-d)pyrimidine) have shown that the African trypanosomes are biologically and biochemically similar to Leishmania spp. and Trypanosoma cruzi with respect to their response to this compound. These organisms, as a group, share the unique ability to convert allopurinol sequentially to its ribonucleoside monophosphate and 4-aminopyrazolo(3,4-d)pyrimidine ribonucleoside mono-, di- and triphosphates.

Concerted inhibition of a NADP+-specific isocitrate dehydrogenase and the implications for metabolic regulation☆

Abstract A possible physiologic role for the concerted inhibition of a NADP + -specific isocitrate dehydrogenase by oxalacetate and glyoxylate is presented. The significance of this inhibition is also discussed with respect to the “induced fit” theory of enzyme-substrate interaction. However, since numerous structural analogues of these two compounds were without effect, it is believed that these two components are not acting as a “synthetic” substrate.

Purine Salvage Enzymes in Man and Leishmania donovani

A comparison of the enzymes of pathogenic protozoa to those of man is of fundamental importance to the search for much needed chemotherapeutic agents. The enzymes involved in purine salvage are of particular interest because most pathogenic protozoa lack the ability to synthesize purines de novo and consequently are obligate salvagers of preformed purines.

The relationship of soluble and mitochondrial isocitrate dehydrogenases in metabolic regulation

Abstract It has been shown that the soluble NADP+-specific isocitrate dehydrogenases from both prokaryotic and eukaryotic cells are inhibited by oxalacetate and glyoxylate in a concerted manner. We have now compared the mitochondrial and soluble isocitrate dehydrogenases from N. crassa and S. cerevisiae with respect to their inhibition by these compounds. The NAD+-linked mitochondrial enzymes were not inhibited, whereas the NADP+-linked enzymes were. The significance of this inhibition is discussed with respect to the regulatory role of these two enzymes.