Subcellular localization of phosphoenolpyruvate carboxykinase in the trypanosomatids Trypanosoma cruzi and Crithidia fasciculata.
Particulate fractions obtained from Trypanosoma cruzi and Crithidia fasciculata by different procedures were subjected to isopycnic centrifugation in sucrose gradients, in order to determine the subcellular localization of phosphoenolpyruvate carboxykinase (PEPCK) in both organisms, and of malic enzyme (ME) I in T. cruzi. The more clear-cut results were obtained with T. cruzi by breaking the cells by grinding in a mortar with silicon carbide and using a gradient from 0.4 to 2.0 M sucrose, whereas with C. fasciculata, the best procedure was disruption of the cells by digitonin treatment and potter homogenization and use of a gradient from 1.1 to 2.0 M sucrose. PEPCK banded together with the glycosomal marker hexokinase in both organisms; there was a clear separation from the mitochondrial markers, oligomycin-sensitive Mg2+-APTase and citrate synthase. PEPCK showed a latency of 24% in the enriched 'glycosoma' fraction of T. cruzi. ME I from T. cruzi, on the other hand, banded together with the mitochondrial markers. These results indicate that PEPCK and ME are present in different subcellular compartments, a fact significant for the prevention of a futile cycle between C4-dicarboxylic acids and C3-monocarboxylic acids, which might take place if both enzymes functioned in the same compartment.
D000818:Animals; D002458:Cell Fractionation; D003421:Crithidia / Q000201:enzymology* / Q000648:ultrastructure; D008291:Malate Dehydrogenase / Q000378:metabolism; D008830:Microbodies / Q000201:enzymology; D008928:Mitochondria / Q000201:enzymology; D010729:Phosphoenolpyruvate Carboxykinase (GTP) / Q000378:metabolism*; D013347:Subcellular Fractions / Q000201:enzymology; D014349:Trypanosoma cruzi / Q000201:enzymology* / Q000648:ultrastructure
D008291:Malate Dehydrogenase; D010729:Phosphoenolpyruvate Carboxykinase (GTP)