Investigation of reconstitution of the Na, K-ATPase in lipid vesicles.

Vesicles containing Na,K-ATPase were prepared by a dialysis method in buffers with various concentrations of K+ and Na+ ions. Ion-exchange chromatography has been used to separate proteoliposomes into protein-depleted and protein-rich fractions. The pumping activity of reconstituted ion pumps has been determined in the different fractions of the vesicle preparation using voltage-dependent fluorescence dyes. This method allowed to characterise vesicle fractions by a quantity which is proportional to the average number of pumps per vesicle with an active (inside-out) orientation. It could be shown that both, the amount of enzymatic active protein and the orientation of Na,K-ATPase in the vesicle lipid bilayer, is partially controlled by the Na+ and K+ concentration in the buffer during vesicle formation. High Na+ concentrations preferentially maintain the E1 conformation of the enzyme, which is less stable against denaturation during the dialysis, but displays a higher percentage of inside-out orientation of the transport-active protein. High K+ concentrations maintain the E2 conformation of the enzyme, which is stable against denaturation during the dialysis, but leads to a random orientation of the pump during dialysis.