Reconstitution of active transport catalyzed by the purified sodium and potassium ion-stimulated adenosine triphosphatase from canine renal medulla.

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+ + I(+)-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 22Naf, dependent on externally added ATP and inhibitable by internally trapped cardiac glycosides, to a level 3.fold higher (60 mbr) than‘ the initial concentration of Na+ within the vesicles (20 m&r). Double label experiments with 4K+ and 22Na+ indicated that the ratio of K+ etliux to Na+ uptake is far below the 2 :3 ratio of K+ influx to Naf efllux observed in nerve axons and erythrocyte ghosts. Parallel experiments employing asC1and 2zNa+ demonstrated that 36C1is 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 Clalong with actively pumped Na+, rather than exchange of K+ for Na+, cannot be explained by the observation that the permeability of the vesicles to Clis roughly Z-fold higher than that for I(+. It appears that this reconstituted (Na+ + I(+)-ATPase either is capable of pumping Clas well as Na+ or is equipped with some specific mechanism for translocating Clalong with actively pumped Na+.