Reconstitution of beef heart mitochondrial F0F1 in reverse phase evaporation vesicles.
Beef heart mitochondrial F0F1 was reconstituted in proteoliposomes by a new procedure. MF0F1 was inserted in preformed reverse phase evaporation vesicles of large diameters prepared from asolectin (MF0F1-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 MF0F1-REV proteoliposomes catalyzed an exchange between Pi and ATP and were capable of proton pumping. Both reactions were inhibited by oligomycin and uncoupler of oxidative phosphorylation. The range of Pi-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 F1 and other coupling factors are not required to obtain high Pi-ATP exchange activity by MF0F1-REV. This simple and rapid reconstitution procedure should be useful for future studies dealing with functional analysis of MF0F1.
Knowles
A F
AF
Department of Biochemistry and Molecular Biology, SUNY Health Science Center, Syracuse, NY 13210, USA. afknowles@worldnet.att.net
Penefsky
H S
HS
D000818:Animals; D002384:Catalysis; D002417:Cattle; D003902:Detergents; D007700:Kinetics; D008081:Liposomes; D008929:Mitochondria, Heart / Q000201:enzymology*; D009840:Oligomycins / Q000494:pharmacology; D010743:Phospholipids; D011510:Proteolipids / Q000737:chemistry* / Q000378:metabolism*; D006180:Proton-Translocating ATPases / Q000737:chemistry* / Q000378:metabolism*; D013329:Structure-Activity Relationship; D014475:Uncoupling Agents / Q000494:pharmacology
D003902:Detergents; D008081:Liposomes; D009840:Oligomycins; D010743:Phospholipids; D011510:Proteolipids; D014475:Uncoupling Agents; C020485:proteoliposomes; D006180:Proton-Translocating ATPases