title: Structural analysis of the products of chymotryptic cleavage of the E1 form of Na,K-ATPase alpha-subunit: identification of the N-terminal fragments containing the transmembrane H1-H2 domain.
abstract: 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.
authors.affiliation: Department of Pharmacology, Medical College of Ohio, Toledo 43614, USA.
authors.forename: A
authors.initials: A
authors.lastname: Ivanov
authors.forename: A
authors.initials: A
authors.lastname: Askari
authors.forename: N N
authors.initials: NN
authors.lastname: Modyanov
mesh_terms: D000818:Animals; D002462:Cell Membrane / Q000201:enzymology; D002918:Chymotrypsin / Q000378:metabolism*; D003432:Cross-Linking Reagents; D004285:Dogs; D007679:Kidney Medulla / Q000201:enzymology; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry*; D010618:Phenanthrolines; D017421:Sequence Analysis; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry*
chemical_list: D003432:Cross-Linking Reagents; D010446:Peptide Fragments; D010618:Phenanthrolines; D002918:Chymotrypsin; D000254:Sodium-Potassium-Exchanging ATPase; C025205:1,10-phenanthroline