TY - JOUR
T1 - Functional characterization and subcellular localization of the three malate dehydrogenase isozymes in Leishmania spp.
AU - Leroux, Alejandro
AU - Fleming-Canepa, Ximena
AU - Aranda, Alejandro
AU - Maugeri, Dante
AU - Cazzulo, Juan J.
AU - Sánchez, Marco A.
AU - Nowicki, Cristina
N1 - Funding Information:
This work was performed with grants from Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Universidad de Buenos Aires (UBA) and Agencia Nacional de Promoción Científica y Tecnológica (ANPCYT), Argentina. CN and JJC are members of the Research Career from CONICET, XFC was supported by a fellowship from ANPCYT, AL and AA are student fellows from Buenos Aires University and DM was supported by a fellowship from UNSAM (Argentina). MAS is supported by award No. 0560056z from the American Heart Association (USA).
PY - 2006/9
Y1 - 2006/9
N2 - 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 (≅57 kDa) 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.
AB - 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 (≅57 kDa) 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.
KW - Leishmania spp.
KW - Malate dehydrogenase isozymes
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U2 - 10.1016/j.molbiopara.2006.04.010
DO - 10.1016/j.molbiopara.2006.04.010
M3 - Article
C2 - 16750864
AN - SCOPUS:33746003944
SN - 0166-6851
VL - 149
SP - 74
EP - 85
JO - Molecular and Biochemical Parasitology
JF - Molecular and Biochemical Parasitology
IS - 1
ER -