Abstract

This report describes the first isolation and molecular characterization of the mitochondrial F1-ATPase from Trypanosoma brucei. The isolation procedure utilized is a modified chloroform extraction procedure. In contrast to earlier reports on the F1-ATPase from other trypanosomatids, the F1-ATPase we have isolated from the procyclic form of T. brucei a complex composed of five distinct subunits. Apparent molecular weights of these subunits are 55,000 [alpha], 42,000 [beta], 32,000 [gamma], 22,000 [delta], and 17,000 [epsilon]. The F1 moiety which possesses the active site of the H(+)-ATPase has an ATPase activity in the standard Tris-HCl coupled enzyme assay with a Vmax of 22.96 mumol min-1 (mg protein)-1 and a Km value of 0.60 mM. This ATPase activity is cold labile and is not susceptible to oligomycin inhibition as is the membrane bound enzyme. Upon reconstitution with F1-ATPase depleted membranes (urea particles) the ATPase regains oligomycin sensitivity to the same extent as that found in the intact inner membrane vesicles. ATP synthesis is also restored to these particles upon reconstitution with F1. These results indicate that this F1-ATPase as isolated is intact with respect to all the critical H(+)-ATPase functions.