title: Conformational transitions of the H,K-ATPase studied with sodium ions as surrogates for protons.
abstract: Following a recent demonstration that H,K-ATPase can active transport Na+ at a low rate (Polvani, C., Sachs, G., and Blostein, R. (1989) J. Biol. Chem. 264, 17854-17859), we have looked for and found effects of Na+ ions on the conformational state of gastric H,K-ATPase labeled with fluorescein isothiocyanate. Na+ ions reverse the K(+)-induced quench of the fluorescein fluorescence and somewhat enhance fluorescence in the absence of K+ ions. Equilibrium titrations of the cation effects show that Na+ and K+ ions are strictly competitive with apparent dissociation constants of KNa+ = 62 mM (n = 2) and KK+ = 6.6 mM (n = 2). The observations demonstrate that Na+ ions bind to and stabilize the high fluorescence E1 form of the protein while K+ ions stabilize the low fluorescence E2 form. Elevation of pH from 6.4 to 8.0 increased the apparent affinity of the Na+ ions from approximately 62 to 10.2 mM, consistent with competition between protons and Na+. The action of Na+ to stabilize the E1 form was used to measure the rate of the E2K----E1Na transition with a stopped-flow fluorimeter. The rate at pH 6.4 and 20 degrees C is 18.1 s-1. In addition the rate of the reverse conformational transition E1K----E2K has been measured at several K+ concentrations. From the hyperbolic dependence on K+ concentration a maximal rate of 211 +/- 32 s-1 and intrinsic K+ dissociation constant on E1 of 64.6 +/- 3.3 mM have been estimated. The kinetic and equilibrium data are self-consistent and thus support the proposed action of Na+ and K+ ions. Compared with Na,K-ATPase, the H,K-ATPase exhibits a lower affinity for Na+ on E1 and a much faster rate of the E2K----E1Na transition, but a similar affinity for K+ ions on E1 and rate of the transition E1K----E2K. The significance of the similarities and differences in cation specificity and rates of conformational changes of Na,K- and H,K-ATPases is discussed.
authors.affiliation: Center for Ulcer Research and Education, Veterans Administration Center, Wadsworth Division, Los Angeles, California 90073.
authors.forename: E C
authors.initials: EC
authors.lastname: Rabon
authors.forename: S
authors.initials: S
authors.lastname: Bassilian
authors.forename: G
authors.initials: G
authors.lastname: Sachs
authors.forename: S J
authors.initials: SJ
authors.lastname: Karlish
mesh_terms: D000251:Adenosine Triphosphatases / Q000737:chemistry* / Q000378:metabolism; D002414:Cations, Monovalent; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins; D005456:Fluorescent Dyes; D017506:H(+)-K(+)-Exchanging ATPase; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008861:Microsomes / Q000201:enzymology; D011188:Potassium / Q000494:pharmacology; D011487:Protein Conformation / Q000187:drug effects; D012964:Sodium / Q000494:pharmacology*; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D013270:Stomach / Q000648:ultrastructure; D013861:Thiocyanates
chemical_list: D002414:Cations, Monovalent; D005452:Fluoresceins; D005456:Fluorescent Dyes; D013861:Thiocyanates; D012964:Sodium; D000251:Adenosine Triphosphatases; D017506:H(+)-K(+)-Exchanging ATPase; D000254:Sodium-Potassium-Exchanging ATPase; D016650:Fluorescein-5-isothiocyanate; D011188:Potassium