title: Nucleotide-binding kinetics of Na,K-ATPase: cation dependence.
abstract: Correlation between the Na,K-ATPase affinity to ADP and the cation (its nature and concentration) present in the medium was investigated. In buffer with low ionic strength (I approximately 1 mM) high-affinity ADP binding was not observed, while a stepwise increase in the concentrations of added cation (Na(+), Tris(+), imidazole(+), N-methylglucamine(+), choline(+)) induced an increase in the ADP affinity. The effect was fully saturated at 30-50 mM for all of the cations tested. The maximal affinity for ADP was slightly higher in the presence of Na(+), Tris(+), or imidazole(+) than in the presence of N-methylglucamine(+) or choline(+) (equilibrium dissociation constant K(d) 0.2-0.3 vs 0.7 microM). The ADP dissociation rates from its complex with enzyme in the presence of Na(+) or Tris(+) were similar, implying identity of the nucleotide-binding enzyme conformations, which therefore are assigned to E(1). The ability to compete with K(+) clearly distinguished Na(+) from other cations, which speaks against the sole involvement of the transport sites in the induction of the ADP-binding E(1) conformation. Since the cations are similar in their mode of induction of the high ADP affinity but they demonstrate a pronounced difference in ability to compete with K(+), their effects cannot be combined within any scheme with only one type of cation-binding sites. We suggest that the high affinity toward nucleotide is induced by cation interactions within the protein or lipid and that these nucleotide-domain-related sites coexist with the transport sites, which bind only Na(+) or K(+).
authors.affiliation: Department of Biophysics, University of Aarhus, Ole Worms Allé 185, DK-8000 Aarhus C, Denmark. nf@biophys.au.dk
authors.forename: Natalya U
authors.initials: NU
authors.lastname: Fedosova
authors.forename: Mikael
authors.initials: M
authors.lastname: Esmann
mesh_terms: D000244:Adenosine Diphosphate / Q000037:antagonists & inhibitors / Q000737:chemistry*; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D001692:Biological Transport; D002414:Cations, Monovalent / Q000737:chemistry*; D002794:Choline / Q000737:chemistry; D007093:Imidazoles / Q000737:chemistry; D007668:Kidney / Q000201:enzymology; D008536:Meglumine / Q000737:chemistry; D011188:Potassium / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D012964:Sodium / Q000737:chemistry; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry*; D013552:Swine; D014325:Tromethamine / Q000737:chemistry
chemical_list: D002414:Cations, Monovalent; D007093:Imidazoles; D014325:Tromethamine; D000244:Adenosine Diphosphate; D008536:Meglumine; C029899:imidazole; D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase; D002794:Choline; D011188:Potassium