Ultrastructure of membrane-bound Na,K-ATPase after extensive tryptic digestion

AbstractMembrane-bound Na,K-ATPase was digested with trypsin in the presence of Rb+ to form the stable 19-kDa and smaller fragments of the α-chain known to preserve occlusion of Rb+ (K+) or Na+. The trypsinized membranes obtained from pig kidney and shark rectal gland were analyzed by electron microscopy. Tryptic digestion preserved general membrane structure but removed both the surface particles observed by negative staining and the protruding cytoplasmic portion of the α-subunit identified in thin sections. However, intramembrane particles defined by freeze-fracture were preserved after trypsinization suggesting that the remaining membrane spanning protein fragments retain the native structure within the lipid bilayer after proteolysis