title: Identification of the cation binding domain of Na/K-ATPase.
abstract: This paper summarises results and conclusions from experiments with renal Na/K-ATPase, utilising proteolytic digestion to define minimal peptide structures involved in cation occlusion and chemical modification with dicyclohexylcarbodiimide (DCCD) to investigate the role of carboxyl groups and location of K (Rb) and/or Na binding residues. Extensive digestion with trypsin or non-selective proteases in the presence of Na or Rb and absence of divalent cations reveals an essential C-terminal 19Kd fragment of the alpha chain (N-terminal asn 830) and indicates that occlusion sites of Na or K ions must reside within transmembrane segments. The bulk of the beta chain is not involved. Kinetics of inactivation of Rb or Na occlusion and covalent labelling with DCCD indicate that each of two Rb(K) or Na sites contains a carboxyl group. The third Na site may contain only neutral ligating groups. One carboxyl group is located on the 19Kd fragment and the other on tryptic fragment of about 9Kd. When cyanogen bromide was used to digest labelled alpha chain, glu 953 was found to be labelled in a Rb-protectable fashion. In tryptic "19Kd-membranes", fragments containing all putative transmembrane segments of the alpha chain have been identified (i.e. 19, 10.9, 8.7 and 8.0 Kda respectively). The cation occlusion "cage" is apparently composed of ligating groups from different trans-membrane segments, including segments of the 19Kd fragment. Construction of models is hampered by the fact that the number of the transmembrane segments is still uncertain, particularly in the crucial C-terminal domain. Alternative ways of arranging the tryptic fragments across the membrane are discussed.
authors.affiliation: Department of Biochemistry, Weizmann Institute of Science, Rehovot, Israel.
authors.forename: S J
authors.initials: SJ
authors.lastname: Karlish
authors.forename: R
authors.initials: R
authors.lastname: Goldshleger
authors.forename: D M
authors.initials: DM
authors.lastname: Tal
authors.forename: J M
authors.initials: JM
authors.lastname: Capasso
authors.forename: S
authors.initials: S
authors.lastname: Hoving
authors.forename: W D
authors.initials: WD
authors.lastname: Stein
mesh_terms: D000818:Animals; D002412:Cations / Q000378:metabolism*; D006801:Humans; D011487:Protein Conformation; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*
chemical_list: D002412:Cations; D000254:Sodium-Potassium-Exchanging ATPase