oligomycins and 3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate

The F0 portion of the rat liver mitochondrial ATP synthase (F0F1-ATPase) has been purified by a rapid, high yield procedure. F0 is selectively extracted from inner membrane vesicles with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) after prior treatment of the vesicles with guanidine HCl to remove F1. The resultant F0 is functional in proton translocation assays and separates in sodium dodecyl sulfate-polyacrylamide gel electrophoresis into four major and three minor Coomassie-stainable bands, all with apparent molecular masses below 30 kDa. This CHAPS-purified F0 preparation was characterized in detail for its capacity to interact with the unique probe diethylstilbestrol (DES) which, depending on conditions, has been shown to interact with rat liver F0F1 to either inhibit or promote ATP hydrolysis (McEnery, M. W., and Pedersen, P.L. (1986) J. Biol. Chem. 261, 1745-1752). DES-inhibitory sensitivity could be conferred on F1-ATPase activity with the same concentration dependence on F0 as conferral of oligomycin sensitivity. DES was shown also to inhibit the magnitude of valinomycin induced proton influx, while initiating proton efflux in asolectin vesicles reconstituted with F0 and loaded with K+. The potency of DES in producing the latter effects was shown to be highly dependent on hydroxyl groups in "para" positions of the two benzene rings within the DES molecule. Finally, in the absence of F0, DES was shown to act as a catalyst of proton influx in K+-loaded asolectin vesicles upon addition of valinomycin. A model based on the structure of DES is presented to account for both the inhibitory and uncoupling properties of this compound.

Topics: Animals; Cholic Acids; Diethylstilbestrol; Electrophoresis, Polyacrylamide Gel; Hydrolysis; Male; Mitochondria, Liver; Oligomycins; Proton-Translocating ATPases; Protons; Rats

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.

Topics: Animals; Cholic Acids; Detergents; Electrophoresis, Polyacrylamide Gel; Kinetics; Liposomes; Male; Microscopy, Electron; Mitochondria, Liver; Oligomycins; Proton-Translocating ATPases; Rats; Solubility