Reconstitution of the proton translocating ATPase from bovine heart mitochondria into planar phospholipid bilayer membranes.
Proton translocating ATPase (F0F1) from bovine heart mitochondria was reconstituted into planar phospholipid bilayers, and its electrogenicity was directly demonstrated. The F0F1 ATPase was solubilized using 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonic acid (CHAPS) as a detergent followed by sucrose density gradient centrifugation according to the method originally described by McEnery et al. for rat liver mitochondria (McEnery et al. (1986) J. Biol. Chem. 259, 4642-4651), with minor modifications. The purified ATPase was reconstituted into proteoliposomes and then reconstituted into planar phospholipid bilayers by the modified fusion method (Hirata et al. (1986) J. Biol. Chem. 261, 9839-9843). A short-circuit current of up to 0.4 pA was induced by adding ATP, and this current was suppressed by the F1 ATPase inhibitor NaN3 or by a specific mitochondrial F0 inhibitor, oligomycin. The direction of the current corresponded to the flow of positive charges from the F1 side to the F0 side. All these facts clearly demonstrate that the mitochondrial F0F1 ATPase was successfully reconstituted into planar phospholipid bilayers, and the current was generated by the ATPase.
D000255:Adenosine Triphosphate / Q000378:metabolism / Q000494:pharmacology; D000817:Anilino Naphthalenesulfonates; D000818:Animals; D002417:Cattle; D002793:Cholic Acids; D004553:Electric Conductivity; D005456:Fluorescent Dyes; D008051:Lipid Bilayers / Q000378:metabolism*; D008081:Liposomes / Q000378:metabolism; D008929:Mitochondria, Heart / Q000201:enzymology*; D010743:Phospholipids / Q000378:metabolism*; D006180:Proton-Translocating ATPases / Q000302:isolation & purification / Q000378:metabolism*
D000817:Anilino Naphthalenesulfonates; D002793:Cholic Acids; D005456:Fluorescent Dyes; D008051:Lipid Bilayers; D008081:Liposomes; D010743:Phospholipids; C027132:1-anilino-8-naphthalenesulfonate; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate