Glycosomal and mitochondrial malate dehydrogenases in epimastigotes of Trypanosoma cruzi.
The degradation of glucose by Trypanosoma cruzi leads to the excretion of succinate. Malate dehydrogenase (MDH) participates in this process by reducing to malate the oxaloacetate synthesized by the glycosomal enzyme, phosphoenolpyruvate carboxykinase. The best coupling for these two sequential reactions would be attained if both enzymes were placed in the same subcellular compartment. The intracellular distribution of the MDH activity in epimastigotes of T. cruzi was studied by two methods. Selective disruption of cellular membranes with increasing concentrations of digitonin, indicated that trypanosomal MDH is particulate. Isopycnic centrifugation in a sucrose gradient of a large granule fraction, obtained by grinding the cells with silicon carbide, showed the presence of two MDH activities: one banding together with the glycosomal marker phosphoenolpyruvate carboxykinase, the other with the mitochondrial marker citrate synthase. Isoelectrofocusing of cell-free extracts led to the separation of two enzyme forms, with pI values of about 3.5 (MDHa) and 9.4 (MDHb). These forms had similar molecular weights (approx. 60 000) and apparent Km values, but showed a small but consistent difference in their pH optima (9.23 for MDHa and 9.05 for MDHb), and in their activation by inorganic phosphate (apparent Ka values of 33 mM and 87 mM, for MDHa and MDHb, respectively). Determination of the pH optima of the enzyme forms separated by isopycnic centrifugation suggests that the glycosomal enzyme form is MDHa, and the mitochondrial one is MDHb.
D000818:Animals; D005947:Glucose / Q000378:metabolism; D007526:Isoelectric Point; D008291:Malate Dehydrogenase / Q000302:isolation & purification / Q000378:metabolism*; D008928:Mitochondria / Q000378:metabolism; D013347:Subcellular Fractions / Q000378:metabolism; D013386:Succinates / Q000096:biosynthesis; D019802:Succinic Acid; D014349:Trypanosoma cruzi / Q000378:metabolism*
D013386:Succinates; D019802:Succinic Acid; D008291:Malate Dehydrogenase; D005947:Glucose