Abstract
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+.
Highlights
Microsomal sodium and potassium ion-stimulated adenosine triphosphatase ((Na+ + K+)-ATPase) was prepared from canine renal medulla by the method of Kyte
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)
Summary
The assay used for determination of activity at all stages of purification of the enzyme prior to co-solubilization with phospholipid and reconstitution is that of Nakao et al [14] as modified by Kyte [6]. The assay used on the solubilized and reconstituted enzyme was a modification of Method D of Lindberg and Ernster [15]. For determination of ATPase activity under optimal conditions, 10 to 50 ~1 of solubilized or reconstituted enzyme were incubated for 15 min at 37” f 0.5” in 0.5 ml of the incubation mixture of Kyte. The sample was quenched with 2.5 ml of 1.25%. The two phases were separated by centrifugation for 5 min at top speed-in a tabletop centrifuge
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