Oxidation of alanine, acetate, glutamate, and succinate by digitonin-permeabilized Leishmania major promastigotes.
Leishmania major promastigotes were treated with digitonin and the rates at which [1-14C]acetate, [1,4-14C]succinate, [1-14C]glutamate, and [U-14C]alanine are oxidized were measured in the presence of suitable cofactors. Acetate was oxidized at the lowest rate of the four substrates examined, even in the presence of added NAD, CoA, ADP and acetyl-CoA synthase. Its rate of oxidation was negligible if the permeabilized 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 intact cells. Its rate of oxidation was strongly inhibited by antimycin A, but that of glutamate was scarcely affected. Fumarate inhibited the rate of oxidation of acetate, glutamate, and succinate, but increased that of alanine. Ca++ inhibited the rates of oxidation of alanine and succinate, but not of acetate or glutamate. Increasing the osmolality by addition of mannitol partially inhibited the rate of oxidation of alanine but had little effect on that of glutamate. These results show that appreciable transaminase activity remains in the permeabilized cells and support earlier data indicating the presence of a branched NAD-to-cytochrome oxidase system. These results also provide preliminary information on the sensitivity of the two branches to Ca++, hyperosmolality, and Krebs cycle intermediates.
Blum
J J
JJ
Department of Cell Biology, Duke University Medical Center, Durham, North Carolina 27710, USA.
D000085:Acetates / Q000378:metabolism*; D000409:Alanine / Q000378:metabolism*; D000818:Animals; D000968:Antimycin A / Q000494:pharmacology; D002250:Carbon Radioisotopes; D002463:Cell Membrane Permeability; D004072:Digitonin; D003576:Electron Transport Complex IV / Q000378:metabolism; D005650:Fumarates / Q000494:pharmacology; D018698:Glutamic Acid / Q000378:metabolism*; D007700:Kinetics; D018320:Leishmania major / Q000378:metabolism*; D009243:NAD / Q000378:metabolism; D010084:Oxidation-Reduction; D013386:Succinates / Q000378:metabolism*
D000085:Acetates; D002250:Carbon Radioisotopes; D005650:Fumarates; D013386:Succinates; D009243:NAD; D018698:Glutamic Acid; D000968:Antimycin A; D003576:Electron Transport Complex IV; D004072:Digitonin; D000409:Alanine