Properties of the V₀V₁ Na⁺-ATPase from Enterococcus hirae and Its V₀ Moiety

Abstract

We report here the large-scale purification of vacuolar (V₀V₁)-type Na⁺-ATPase from Enterococcus hirae achieved using column anion-exchange and gel filtration chromatographies; 32 mg of purified enzyme comprising nine subunits, 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 activity of ATP hydrolysis (35.7 μmol P₁ released/min/mg protein). ATP-driven ²²Na⁺ uptake by reconstituted V₀V₁-proteoliposomes exhibited an apparent K_t value for Na⁺ of 40 μM, which is near the K_m value (20 μM) for Na⁺ of the ATP hydrolytic activity. Denatured gel electrophoresis revealed that six subunits, A, B, C, D, E, and F, are releasable as the V₁ subunit from the V₀V₁ complex by incubation with ethylenediaminetetraacetic acid; subunit G was not identified. The remaining V₀-liposomes containing I and K subunits catalyzed Na⁺ uptake in response to potassium diffusion potential (Δψ, inside negative); the K_t value for Na⁺ of this reaction was estimated to be about 2mM. Inhibition by N, N'-dicyclohexylcarbodiimide (DCCD) of the Na⁺-ATPase activity and Δψ-driven Na⁺ uptake by the V₀-liposomes was prevented by the presence of Na⁺, suggesting that the Na⁺ binding site overlaps with the DCCD-reactive site.