Purification and Characterization of the Soluble F(1)-ATPase of Oat Root Mitochondria.

The properties of the soluble moiety (F(1)) of the mitochondrial H(+)-ATPase from oat roots were examined and compared to those of the native mitochondrial membrane-bound enzyme. The chloroform soluble preparation was purified by Sephadex G-200 and DEAE-cellulose chromatography. The purified F(1) preparation contained major polypeptides corresponding to alpha, beta, gamma, delta, and epsilon of apparent molecular mass 58, 55, 35, 22, and 14 kilodaltons, respectively. The purified F(1)-ATPase, like the native enzyme, was inhibited by azide (I(50) = 10 micromolar), nitrate (I(50) = 7-10 millimolar), 4,4'-diisothiocyano-2,2'-stilbene disulfonic acid (I(50) = 1-3 micromolar), and 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (I(50) = 3 micromolar). F(1)-ATPase activity was stimulated by bicarbonate but not by chloride. In both the native and the F(1)-form of the ATPase, ATP was hydrolyzed in preference to GTP. The results indicate that these properties of the native membrane-bound mitochondrial ATPase have been conserved in the purified F(1). In contrast to the membrane-bound enzyme, the F(1)-ATPase was not inhibited by oligomycin or by N,N'-dicyclohexylcarbodiimide. The mitochondrial F(1)-ATPase from oat roots is analogous to other known F(1)F(0)-ATPases.