Improvement of reconstitution of the Cl^--translocating ATPase isolated from Acetabularia acetabulum into liposomes and several anion pump characteristics

The improved reconstitution of the Mono Q-III fraction, a Cl^--translocating ATPase, isolated from Acetabularia acetabulum (Ikeda et al. (1990) Biochemistry 29, 2057-2065) into liposomes rendered transport properties of this enzyme clear. The liposomes were prepared by the reversed-phase method using egg lecithin and cholesterol in a molar ration of 2:1 and the purified ATPase was incorporated into the liposomes by a dialysis for 3 h. About 80% of the ATPase was incorporated into the liposomes. The weight ratio of the enzyme to lipid was 1:400-600. A sigmoid curve was obtained when the Cl^--transport activity of the enzyme was plotted against Cl^- concentration. Hill's plot afforded a half-subsrate concentration [S]_0.5 of 45 mM and a Hill's coefficient n of 2.33. Effects of Br^- and F^- on the Cl^--transport were also examined in the reconstituted system, both halide ions decrease the ³⁶Cl^- efflux significantly. These kinetic data are in good agreement with the electrophysiological data presented by Tittor et al. (1983) J. Membr. Biol. 75, 129-139).