Purification and Properties of the F 1 F o ATPase of Ilyobacter tartaricus , a Sodium Ion Pump

Abstract

ABSTRACT The ATPase of Ilyobacter tartaricus was solubilized from the bacterial membranes and purified. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme revealed the usual subunit pattern of a bacterial F 1 F o ATPase. The polypeptides with apparent molecular masses of 56, 52, 35, 16.5, and 6.5 kDa were identified as the α, β, γ, ɛ, and c subunits, respectively, by N-terminal protein sequencing and comparison with the sequences of the corresponding subunits from the Na + -translocating ATPase of Propionigenium modestum . Two overlapping sequences were obtained for the polypeptides moving with an apparent molecular mass of 22 kDa (tentatively assigned as b and δ subunits). No sequence could be determined for the putative a subunit (apparent molecular mass, 25 kDa). The c subunits formed a strong aggregate with the apparent molecular mass of 50 kDa which required treatment with trichloroacetic acid for dissociation. The ATPase was inhibited by dicyclohexyl carbodiimide, and Na + ions protected the enzyme from this inhibition. The ATPase was specifically activated by Na + or Li + ions, markedly at high pH. After reconstitution into proteoliposomes, the enzyme catalyzed the ATP-dependent transport of Na + , Li + , or H + . Proton transport was specifically inhibited by Na + or Li + ions, indicating a competition between these alkali ions and protons for binding and translocation across the membrane. These experiments characterize the I. tartaricus ATPase as a new member of the family of FS-ATPases, which use Na + as the physiological coupling ion for ATP synthesis.