Journal
MOLECULAR AND BIOCHEMICAL PARASITOLOGY
Volume 161, Issue 1, Pages 12-20Publisher
ELSEVIER SCIENCE BV
DOI: 10.1016/j.molbiopara.2008.05.005
Keywords
aspartate aminotransferases; malate dehydrogenases; Trypanosoma cruzi; Tryponosoma brucei
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Funding
- Consejo Nacional de Investigaciones Cientificas y Tecnicas (CONICET)
- Universidad de Buenos Aires (UBA)
- Agencia Nacional de Promocion Cientifica y Tecnologica (Argentina)
- CONICET
- Universidad Nacional de General San Martin
- Belgian Government
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Three genes encoding putative aspartate aminotransferases (ASATs) were identified in the Tryponosoma cruzi genome. Two of these ASAT genes, presumably corresponding to a cytosolic and mitochondrial isoform, were cloned and expressed as soluble His-tagged proteins in Escherichia coli. The specific activities determined for both T cruzi isozymes were notably higher than the values previously reported for Trypanosoma brucei orthologues. To confirm these differences, T brucei mASAT and cASAT were also expressed as His-tagged enzymes. The kinetic analysis showed that the catalytic parameters of the new recombinant T brucei ASATs were very similar to those determined for T cruzi orthologues. The cASATs from both parasites displayed equally broad substrate specificities, while mASATs were highly specific towards aspartate/2-oxoglutarate. The subcellular localization of the mASAT was confirmed by digitonin extraction of intact epimastigotes. At the protein level, cASAT is constitutively expressed in T. brucei, whereas mASAT is down-regulated in the bloodstream forms. By contrast in T cruzi, mASAT is expressed along the whole life cycle, whereas cASAT is specifically induced in the mammalian stages. Similarly, the expression of malate dehydrogenases (MDHs) is developmentally regulated in T cruzi: while glycosomal MDH is only expressed in epimastigotes and mitochondrial MDH is present in the insect and mammalian stages. Taken together, these findings provide evidence for a metabolically active mitochondrion in the mammalian stages of T cruzi, and suggest that the succinate excreted by amastigotes. More likely represents a side product of an at least partially operative Krebs cycle, than an end product of glycosomal catabolism. (C) 2008 Elsevier B.V. All rights reserved.
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