, Volume 28, Issue 6, pp 459-469

Reconstituted Cl pump protein: A novel ion(Cl)-motive ATPase

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Abstract

Cl absorption by theAplysia californica foregut is effected through an active Cl transport mechanism located in the basolateral membrane of the epithelial absorptive cells. These basolateral membranes contain both Cl-stimulated ATPase and ATP-dependent Cl transport activities which can be incorporated into liposomes via reconstitution. Utilizing the proteoliposomal preparation, it was demonstrated that ATP, and its subsequent hydrolysis, Mg2+, Cl, and a pH optimum of 7.8 were required to generate maximal intraliposomal Cl accumulation, electrical negativity, and ATPase activity. Additionally, an inwardly-directed valinomycininduced K+ diffusion potential, making the liposome interior electrically positive, enhanced both ATP-driven Cl accumulation and electrical potential while an outwardly-directed valinomycininduced K+ diffusion potential, making the liposome interior electrically negative, decreased both ATP-driven Cl accumulation and electrical potential compared with proteoliposomes lacking the ionophore. Either orthovanadate orp-chloromercurobenzene sulfonate inhibited both the ATP-dependent intraliposomal Cl accumulation, intraliposomal negative potential difference, and also Cl-stimulated ATPase activity. Both aspects of Cl pump transport kinetics and its associated catalytic component kinetics were the first obtained utilizing a reconstituted transporter protein. These results strongly support the hypothesis that Cl-ATPase actively transports Cl by an electrogenic process.