Reconstitution of Active Transport Catalyzed by the Purified Sodium and Potassium Ion-stimulated Adenosine Triphosphatase from Canine Renal Medulla

Abstract Microsomal sodium and potassium ion-stimulated adenosine triphosphatase ((Na+ + K+)-ATPase) was prepared from canine renal medulla by the method of Kyte. The two polypeptides of the enzyme were co-purified to homogeneity by solubilization with sodium cholate in the presence of egg lecithin and by removal of contaminating protein by sedimentation. This purified (Na+ + K+)-ATPase was reconstituted into lipid vesicles by slow removal of the cholate. A fraction of this enzyme was oriented in the vesicle membranes in such a way as to catalyze active uptake of 22Na+, dependent on externally added ATP and inhibitable by internally trapped cardiac glycosides, to a level 3-fold higher (60 mm) than the initial concentration of Na+ within the vesicles (20 mm). Double label experiments with 42K+ and 22Na+ indicated that the ratio of K+ efflux to Na+ uptake is far below the 2:3 ratio of K+ influx to Na+ efflux observed in nerve axons and erythrocyte ghosts. Parallel experiments employing 36Cl- and 22Na+ demonstrated that 36Cl- is co-transported along with 22Na+ in the amount necessary to maintain bulk electrical neutrality of charge transported across the membrane. The mechanism of selective transport of Cl- along with actively pumped Na+, rather than exchange of K+ for Na+, cannot be explained by the observation that the permeability of the vesicles to Cl- is roughly 2-fold higher than that for K+. It appears that this reconstituted (Na+ + K+)-ATPase either is capable of pumping Cl- as well as Na+ or is equipped with some specific mechanism for translocating Cl- along with actively pumped Na+.