title: Energy-transducing H+-ATPase of Escherichia coli. Reconstitution of proton translocation activity of the intrinsic membrane sector.
abstract: The intrinsic membrane sector (Fo) of the H+-ATPase complex of Escherichia coli has been purified, incorporated into liposomes, and its proton-translocating activity reconstituted. The Fo sector was prepared by treating a purified, particulate, F1FO-ATPase preparation with EDTA to solubilize the F1-ATPase. The resulting particulate Fo fraction was incorporated into liposomes of E. coli phospholipids by sonication. Proton efflux from these liposomes was measured with a pH electrode after imposition of a membrane potential. The kinetics of proton efflux fits that predicted by the Goldman-flux equation. The rate of proton efflux was increased maximally more than 100-fold on incorporation of the Fo sector into the liposomes. The rate of H+ efflux varied directly with the amount of Fo material added during reconstitution. Dicyclohexylcarbodiimide blocked Fo-mediated H+ efflux. Inhibition was shown to be due to reaction of dicyclohexylcarbodiimide with a specific proteolipid subunit of Fo. The preparation of Fo used in these studies contained the three proteins that had previously been identified as likely subunits of Fo (Foster, D. L., and Fillingame, R. H. (1979) J. Biol. Chem. 254, 8230-8236). It remains to be determined whether all three components are required for reconstitution of proton translocation activity.
mesh_terms: D000251:Adenosine Triphosphatases / Q000302:isolation & purification / Q000378:metabolism*; D001693:Biological Transport, Active / Q000187:drug effects; D002462:Cell Membrane / Q000378:metabolism; D004024:Dicyclohexylcarbodiimide / Q000494:pharmacology; D004926:Escherichia coli / Q000187:drug effects / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008081:Liposomes; D008433:Mathematics; D006180:Proton-Translocating ATPases
chemical_list: D008081:Liposomes; D004024:Dicyclohexylcarbodiimide; D000251:Adenosine Triphosphatases; D006180:Proton-Translocating ATPases