Purification and proteolysis of vesicles containing inside-out and right-side-out oriented reconstituted (Na+,...

The (Na+,K+)-ATPase from dog kidney has been reconstituted into egg lecithin vesicles (Goldin, S. M. (1977) J. Biol. Chem. 252, 5630-5642). Using sucrose density gradient centrifugation, we have isolated sealed vesicle populations in which the protein molecules have defined orientations. Sealed vesicles sedimented at higher density than unsealed vesicles after equilibration with CsCl. Vesicles containing inside-out oriented enzyme sedimented at lower density than vesicles containing right-side-out oriented enzyme after the internally trapped Cs+ had been pumped out during an incubation with Mg2+ and ATP. Pools of gradient fractions representing unsealed vesicles and sealed vesicles containing inside-out and right-side-out oriented protein were characterized with respect to orientation and degree of sealing by determination of the ATPase activity, the rate of ATP-dependent Na+ uptake, and the inhibition of ATPase activity by ouabain. The accessibilities of sialic acid and of a tryptic site in the vesicle populations were in agreement with the proposed orientations of the protein. The structure of the reconstituted (Na+,K+)-ATPase was examined by proteolysis with trypsin and chymotrypsin over a range of reconstitution protocols. The fragmentation patterns demonstrate that the cholate-reconstituted enzyme, although functionally competent, differs in structure from the native purified enzyme.