IQUIFIB   02644
INSTITUTO DE QUIMICA Y FISICOQUIMICA BIOLOGICAS "PROF. ALEJANDRO C. PALADINI"
Unidad Ejecutora - UE
artículos
Título:
Functional characterization and subcelullar localization of the three malate dehydrogenase isozymes in Leishmania spp
Autor/es:
ALEJANDRO LEROUX; XIMENA FLEMING-CANEPA; ALEJANDRO ARANDA; DANTE MAUGERI; JUAN JOSÉ CAZZULO; MARCO SANCHEZ; CRISTINA NOWICKI
Revista:
MOLECULAR AND BIOCHEMICAL PARASITOLOGY
Editorial:
Elsevier
Referencias:
Año: 2006 vol. 149 p. 74 - 85
ISSN:
0166-6851
Resumen:
As part of a study on the malate dehydrogenase isozymes (MDHs) from Trypanosomatids, three different fractions with MDH activity were obtained from crude extracts of Leishmania mexicana promastigotes combining two different chromatographic steps. Gel filtration chromatography in native conditions showed that most of the MDH activity present in the crude extracts eluted in a single peak, which corresponded to a lower apparent molecular mass ( congruent with 57kDa) than the value expected for typical MDHs. To further characterize the leishmanial isozymes, three putative MDH genes, presumably corresponding to the mitochondrial, glycosomal and cytosolic isoforms were amplified by PCR, cloned into bacterial expression vectors, and the recombinant enzymes purified. Digitonin extraction of intact L. mexicana promastigotes and immunofluorescence microscopy of L. major promastigotes confirmed the subcellular compartmentation of each of the three isozymes. Western blot analysis showed that the three MDHs are developmentally regulated. At the protein level, these isozymes are remarkably more abundant in amastigotes than in promastigotes of L. mexicana. Altogether our results demonstrate the presence of three MDH isoforms with slightly distinct biochemical properties and different subcellular localization in Leishmania spp. Presumably the functional and biochemical features of these isozymes reflect the metabolic adaptation to the different nutrient sources these parasites have to face along their life cycle. These results also emphasize the differences among Trypanosomatids in this area of metabolism, since in the case of Trypanosoma brucei the cMDH is the only isoform expressed in bloodstream trypomastigotes, whereas in Trypanosoma cruzi cMDH is absent. PMID: 16750864 [PubMed - indexed for MEDLINE]