RECONSTITUTION OF BEEF-HEART MITOCHONDRIAL F0F1 IN REVERSE-PHASE EVAPORATION VESICLES

Beef heart mitochondrial FoF1 was reconstituted in proteoliposomes by a new procedure. MFoF1 was inserted in preformed reverse phase evapora tion vesicles of large diameters prepared from asolectin (MFoF1-REV). Reconstitution was mediated by Triton X-100, which was subsequently re moved by treatment with Bio-Beads. Parameters which resulted in optima l reconstitution were described. The MFoF1-REV proteoliposomes catalyz ed an exchange between P-i and ATP and were capable of proton pumping. Both reactions were inhibited by oligomycin and uncoupler of oxidativ e phosphorylation. The range of P-i-ATP exchange activity of the prote oliposomes (70-110 nmol min(-1) mg(-1)) compared favorably with activi ties obtained in vesicles reconstituted by cholate dialysis or cholate dilution. The most important aspect of this method is that, unlike ot her reconstitution methods, exogenous F-1 and other coupling factors a re not required to obtain high P-i-ATP exchange activity by MFoF1-REV. This simple and rapid reconstitution procedure should be useful for f uture studies dealing with functional analysis of MFoF1. (C) 1997 Else vier Science B.V.