Structural analysis of the products of chymotryptic cleavage of the E1 form of Na,K‐ATPase α‐subunit: identification of the N‐terminal fragments containing the transmembrane H1‐H2 domain

Chymotryptic cleavage of the Na,K-ATPase in NaCl medium abolishes ATPase activity and alters other functional parameters. The structure of this modified enzyme is uncertain since only one product of selective proteolysis, the 83-kDa fragment of the alpha-subunit (Ala267-C-terminus) has been identified previously. Here, we applied additional tryptic digestion followed by oxidative cross-linking to identify the products originating from the N-terminal part of the alpha-subunit. These fragments start at Ala72 or Thr74 and contain the transmembrane H1-H2 domain. Formation of cross-linked product between alpha-fragments containing H1-H2 and H7-H10 demonstrate that the structural integrity of the membrane moiety is preserved. We also determined that secondary cleavage of the 83-kDa fragment leads to the formation of C-terminal 48-kDa alpha-fragments with multiple N-termini at Ile582, Ser583, Met584 and Ile585.