Topological localization of proteolytic sites of sodium and potassium ion-activated adenosine triphosphatase

Abstract

Na+,K+-ATPase consists of 2 different polypeptides, .alpha. and .beta., both of which are embedded in the plasma membrane. The .alpha. chain from dog kidney (Na+,K+)-ATPase can be hydrolyzed at specific sites by trypsin and chymotrypsin [Castro, et Farley (1979)]. In order to position these sites with respect to the lipid bilayer, sealed, inside out veiscles from human red cells and unsealed kidney enzyme membranes were treated with trypsin and chymotrypsin; ouabain-stimulated phosphorylation was used to identify the (Na+,K+)-ATPase and its fragments. All of the proteolytic sites observed in the kidney membranes are accessible in the inside-out vesicles. The ouabain-inhibitable uptake of 86Rb+ in human red blood cells is resistant to externally added chymotrypsin. Apparently, the proteolytic sites of the (Na+,K+)-ATPase are exposed on the cytoplasmic side of the membrane.