OXIDATION OF ALANINE, ACETATE, GLUTAMATE, AND SUCCINATE BY DIGITONIN-PERMEABILIZED LEISHMANIA-MAJOR PROMASTIGOTES

Leishmania major promastigotes were treated with digitonin and the rat es at which [1-C-14]acetate, [1,4-C-14]succinate, [1-C-14]glutamate, a nd [U-C-14]alanine are oxidized were measured in the presence of suita ble cofactors. Acetate was oxidized at the lowest rate of the four sub strates examined, even in the presence of added NAD, CoA, ADP and acet yl-CoA synthase. Its rate of oxidation was negligible if the permeabil ized cells were washed before the cofactors were added, indicating the requirement for an as yet unknown factor. Succinate was oxidized at a rate much higher than the very slow rate at which it is oxidized by i ntact cells. Its rate of oxidation was strongly inhibited by antimycin A, but that of glutamate was scarcely affected. Fumarate inhibited th e rate of oxidation of acetate, glutamate, and succinate, but increase d that of alanine. Ca++ inhibited the rates of oxidation of alanine an d succinate, but not of acetate or glutamate. Increasing the osmolalit y by addition of mannitol partially inhibited the rate of oxidation of alanine but had little effect on that of glutamate. These results sho w that appreciable transaminase activity remains in the permeabilized cells and support earlier data indicating the presence of a branched N AD-to-cytochrome oxidase system. These results also provide preliminar y information on the sensitivity of the two branches to Ca++, hyperosm olality, and Krebs cycle intermediates.