alpha-chymotrypsin and beta-aspartyl-phosphate

In this study we examined the coupling of transitions between phosphoforms, E1P and E2P, of pure Na, K-ATPase to cation translocation after selective proteolysis of the alpha-subunit. Cleavage with trypsin at the carboxyl terminal side (Bond 1) of the aspartyl phosphate residue or with chymotrypsin at the aminoterminal side (Bond 1) blocks Na, K-ATPase and Na, K-transport, but the two splits have widely different effects on partial reactions. Cleavage of bond 3 blocks transition from E1P to E2P and abolish both (ADP + ATP)-Na/Na exchange and (ATP + Pi)-Rb/Rb exchange reactions in vesicles reconstituted with pure Na, K-ATPase. Cleavage of bond 1 interferes neither with the transitions nor with the exchange reactions. The results agree with the notion that the transitions between the phosphoforms, E1P and E2P, of the alpha-subunit are coupled to flipping of cation sites between inside exposed (E1) and an outside exposed states (E2).

Topics: Animals; Aspartic Acid; Biological Transport, Active; Chymotrypsin; Kinetics; Lipid Bilayers; Macromolecular Substances; Molecular Weight; Peptide Fragments; Phosphorylation; Potassium; Protein Conformation; Rubidium; Sodium; Sodium-Potassium-Exchanging ATPase; Trypsin