Proton ATPase of rat liver mitochondria. Preparation and visualization of a functional complex using the novel zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate.
The proton ATPase of rat liver mitochondria has been purified by a simple procedure which involves the use of the novel, zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate to solubilize the membrane-bound complex. The purified enzyme has a high, oligomycin-sensitive ATPase activity (11.3 +/- 2.9 mumol/min/mg) in the absence of added phospholipids. It shows, in four different gel electrophoretic systems, the five bands characteristic of the F1 portion of the complex and three additional Coomassie blue-stainable bands which have apparent molecular weights of 28,000, 19,000, and 13,600. A fourth Coomassie blue-stainable component of about 10,000-12,500 daltons comigrates with the delta subunit, whereas a fifth component, detectable only by absorption at 280 nm, is observed between the dye front and the 10,000-dalton species. The enzyme complex has been reconstituted into liposomal vesicles of asolectin. Under these conditions the enzyme catalyzes an ATP-Pi exchange reaction and is capable of translocating protons in an ATP-dependent manner as assayed by quenching of 9-amino-6-chloro-2-methoxyacridine. Both activities are inhibited by the addition of oligomycin, uncoupler, dicyclohexylcarbodiimide, and cadmium. At high detergent concentration, the complex appears in negative stain electron microscopy in a dispersed state. The tripartite structure is clearly visible in monomeric, dimeric, or trimeric forms of the molecule. At the low detergent concentration, the proton ATPase tends to cluster into densely packed arrays. This represents the first report of the properties of a functionally active proton ATPase solubilized and purified in the presence of a zwitterionic detergent.
D000818:Animals; D002793:Cholic Acids; D003902:Detergents; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D008081:Liposomes; D008297:Male; D008854:Microscopy, Electron; D008930:Mitochondria, Liver / Q000201:enzymology*; D009840:Oligomycins / Q000494:pharmacology; D006180:Proton-Translocating ATPases / Q000378:metabolism*; D051381:Rats; D012995:Solubility
D002793:Cholic Acids; D003902:Detergents; D008081:Liposomes; D009840:Oligomycins; D006180:Proton-Translocating ATPases; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate