Adenosine triphosphatase of rat liver mitochondria: detergent solubilization of an oligomycin- and dicyclohexylcarbodiimide-sensitive form of the enzyme

Abstract

The hydrolytic activity of the ATPase [EC 3.6.1.3] bound to purified inner membrane vesicles of rat liver mitochondria can be increased 3-fold by washing extensively with a high ionic strength phosphate buffer. The specific ATPase activities of such phosphate-washed membranes are the highest reported to date for a mitochondrial membrane preparation (21-24 .mu.mol of ATP hydrolyzed min-1 mg-1 in bicarbonate buffer at 37.degree. C). Deoxycholate (0.1 mg/mg of protein) extracts from these membranes a soluble, cold-stable ATPase complex which exhibits a specific activity, under optimal assay conditions, of 12 .mu.mol of ATP hydrolyzed min-1 mg-1. This complex is not sedimented by centrifugation at 201,000 g for 90 min, and readily passes through a 250-.ANG. Millipore filter. The ATPase activity of the soluble complex is inhibited 95% by 2.4 .mu.M oligomycin. In addition, inhibition of 60% or better are obtained in the presence of 1-8 .mu.M dicyclohexylcarbodiimide, p-chloromercuribenzoate, venturicidin, and aurovertin. While a similar complex may be extracted with Triton X-100, this preparation is always lower in both specific activity and in inhibitor sensitivities than the complex extracted with deoxycholate. Detergents of the Tween and Brij series and other detergents of the Triton series are also much less effective than deoxycholate in solubilizing the oligomycin-sensitive ATPase complex of rat liver. The complex extracted with deoxycholate possesses a closer similarity to the membrane-associated ATPase than does the complex extracted with Triton X-100.