Properties of the V0V1 Na+-ATPase from Enterococcus hirae and its V-0 moiety

We report here the large-scale purification of vacuolar (V0V1)-type Na+-ATP ase from Enterococcus hirae achieved using column anion-exchange and gel fi ltration chromatographies; 32 mg of purified enzyme comprising nine subunit s, A, B, C, D, E, F, G, I, and K, was obtained from 20 liter culture. This amount is 500-fold larger than that reported in the previous paper [Murata, T., Takase, K., Yamato, I., Igarashi, K., and Kakinuma, Y. (1997) J. Biol. Chem. 272, 24885-24890]. The purified enzyme shows a high specific activit y of ATP hydrolysis (35.7 mu mol P-i released/min/mg protein). ATP-driven N a-22(+) uptake by reconstituted V0V1-proteoliposomes exhibited an apparent K-t value for Na+ of 40 mu M, which is near the K, value (20 mu M) for Naof the ATP hydrolytic activity. Denatured gel electrophoresis revealed that six subunits, A, B, C, D, E, and F, are releasable as the V-1 subunit from the V0V1 complex by incubation with ethylenediaminetetraacetic acid; subun it Gr was not identified. The remaining V-0-liposomes containing I and K su bunits catalyzed Na+ uptake in response to potassium diffusion potential (D elta psi, inside negative); the K-t value for Na+ of this reaction was esti mated to be about 2 mM. Inhibition by N,N'-dicyclohexyl-carbodiimide (DCCD) of the Na+-ATPase activity and Delta psi-driven Na+ uptake by the V-0-lipo somes was prevented by the presence of Na+, suggesting that the Na+ binding site overlaps with the DCCD-reactive site.