Reconstitution of beef heart mitochondrial F 0F 1 in reverse phase evaporation vesicles

Abstract

Beef heart mitochondrial F 0F 1 was reconstituted in proteoliposomes by a new procedure. MF 0F 1 was inserted in preformed reverse phase evaporation vesicles of large diameters prepared from asolectin (MF 0F 1–REV). Reconstitution was mediated by Triton X-100, which was subsequently removed by treatment with Bio-Beads. Parameters which resulted in optimal reconstitution were described. The MF 0F 1–REV proteoliposomes catalyzed an exchange between P i and ATP and were capable of proton pumping. Both reactions were inhibited by oligomycin and uncoupler of oxidative phosphorylation. The range of P i–ATP exchange activity of the proteoliposomes (70–110 nmol min −1 mg −1) compared favorably with activities obtained in vesicles reconstituted by cholate dialysis or cholate dilution. The most important aspect of this method is that, unlike other reconstitution methods, exogenous F 1 and other coupling factors are not required to obtain high P i–ATP exchange activity by MF 0F 1–REV. This simple and rapid reconstitution procedure should be useful for future studies dealing with functional analysis of MF 0F 1.