Topological localization of proteolytic sites of sodium and potassium ion stimulated adenosinetriphosphatase.
The (Na+ and K+)-stimulated adenosinetriphosphatase [(Na+,K+)-ATPase] consists of two 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, J., & Farley, R. A. (1979) J. Biol. Chem. 254, 2221-2228]. In order to position these sites with respect to the lipid bilayer, we have treated sealed, inside out vesicles from human red cells and unsealed kidney enzyme membranes with trypsin and chymotrypsin and have used ouabain-stimulated phosphorylation 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. These results indicate that the proteolytic sites of the (Na+,K+)-ATPase are exposed on the cytoplasmic side of the membrane.
D000818:Animals; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002918:Chymotrypsin / Q000378:metabolism; D004285:Dogs; D004912:Erythrocytes / Q000201:enzymology; D006801:Humans; D007668:Kidney / Q000201:enzymology; D046911:Macromolecular Substances; D010042:Ouabain / Q000494:pharmacology; D012413:Rubidium / Q000097:blood; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*; D014357:Trypsin / Q000378:metabolism
D046911:Macromolecular Substances; D010042:Ouabain; D002918:Chymotrypsin; D014357:Trypsin; D000254:Sodium-Potassium-Exchanging ATPase; D012413:Rubidium