title: 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 micromol Pi released/min/mg protein). ATP-driven 22Na+ uptake by reconstituted V0V1-proteoliposomes exhibited an apparent Kt value for Na+ of 40 microM, which is near the Km value (20 microM) 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 (Deltapsi, inside negative); the Kt value for Na+ of this reaction was estimated to be about 2 mM. Inhibition by N,N'-dicyclohexylcarbodiimide (DCCD) of the Na+-ATPase activity and Deltapsi-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.
authors.affiliation: Department of Biological Science and Technology, Science University of Tokyo, Noda, Chiba, 278-8510, Japan.
authors.forename: T
authors.initials: T
authors.lastname: Murata
authors.forename: K
authors.initials: K
authors.lastname: Takase
authors.forename: I
authors.initials: I
authors.lastname: Yamato
authors.forename: K
authors.initials: K
authors.lastname: Igarashi
authors.forename: Y
authors.initials: Y
authors.lastname: Kakinuma
mesh_terms: D004024:Dicyclohexylcarbodiimide / Q000494:pharmacology; D016983:Enterococcus / Q000201:enzymology*; D017136:Ion Transport / Q000187:drug effects; D011510:Proteolipids / Q000187:drug effects / Q000378:metabolism; D006180:Proton-Translocating ATPases / Q000302:isolation & purification / Q000378:metabolism*; D012964:Sodium / Q000378:metabolism; D025262:Vacuolar Proton-Translocating ATPases