A comparative study of Leishmania mexicana amastigotes and promastigotes. Enzyme activities and subcellular locations.

Leishmania mexicana mexicana amastigotes have been shown to contain greater activities than promastigotes of the enzymes that catalyse the beta-oxidation of fatty acids, but lower activities of several glycolytic enzymes, with the activity of pyruvate kinase being especially low. The results suggest the beta-oxidation of fatty acids is relatively more important to Leishmania amastigotes than promastigotes, whereas the reverse is true for glycolysis. Succinic dehydrogenase and peptidase activities were much higher in promastigotes than amastigotes. The activities of glucose-6-phosphatase, fructose-1,6-bisphosphatase, acid phosphatase and glucose-6-phosphate dehydrogenase varied less, although in each case the activity was significantly lower in the mammalian stage. A method for lysing and fractionating L. m. mexicana promastigotes has been developed. Using this procedure it has been established that many of the glycolytic and functionally related enzymes are located in cell organelles, that hexokinase is intimately connected with the particulate part of the parasite, and that the microsomal fraction of L. m. mexicana is very different in composition from the microsomes of mammalian liver cells.

The proton gradient across the vacuo-lysosomal membrane of lutoids from the latex of Hevea brasiliensis. I. Further evidence for a proton-translocating ATPase on the vacuo-lysosomal membrane of intact lutoids.

Lutoids (vacuo-lysosomal particles) were isolated from the latex of Hevea brasiliensis. Using flow dialysis with 14C-methylamine uptake as a delta pH probe and 86Rb rubidium + valinomycin distribution for estimations of transmembrane electrical potential, intact lutoids exhibited a delta pH of 1 unit (interior more acid) and a delta psi of -70 mV (interior negative), when suspended in an isotonic medium at physiological concentrations of potassium (30 mM) and pH 7.0, in the absence of ATP. In most cases, the Donnan potential was shown to fully account for delta pH in nonenergized lutoids. The addition of MG-ATP (5 mM) resulted in a marked acidification of the lutoidic internal space (0.7 to 1 pH unit) depending on the composition of the medium, and in a membrane depolarization by 60 mV (interior becoming less negative). The resulting electrochemical potential of protons (delta approximately microH) increased by a hundred millivolts when lutoids were energized by ATP. These data strongly support an inward electrogenic proton translocating function for the ATPase of the vacuo-lysosomal membrane of lutoids. Results are discussed in terms of the in vivo maintenance of large "lutoids/cytoplasm" proton gradients, and of the rôle of these vacuo-lysosomes in the homeostasis of the cytoplasmic metabolism.