title: Partial reactions of the Na,K-ATPase: kinetic analysis and transport properties.
abstract: The complex functions of the Na,K-ATPase can be described by reaction cycles based on the generally accepted "Post-Albers cycle". By appropriate experimental conditions various, partly overlapping partial reactions may be isolated which allow the investigation of specific reaction steps and their succession. From kinetic analysis rate constants and dielectric properties may be determined which characterize the function of the ion pump and allow the formulation of constraints with respect to structure-function relations. This is exemplified by two partial reactions which comprise (1) the ATP-driven Na+ transport, and (2) binding of Na+ ions to the cytoplasm sites. Equilibrium Na+ titration experiments were performed using the fluorescent dyes RH421 and FITC. Fluorescence changes upon addition of Na+ in the presence of various Mg2+ concentrations were similar and the half-saturation concentrations determined were almost identical. As RH421 responds to binding of Na+ to the neutral site whereas FITC monitors conformational changes, this result implies that electrogenic biding of the third Na+ is a trigger for a structural rearrangement of the ATP-binding moiety. This enables enzyme phosphorylation, which is accompanied with a fast occlusion of the Na+ ions and followed by the conformational transition E1/E2 of the protein. Current transients produced by the Na,K-ATPase could be induced by ATP-concentration jumps using DMB-caged ATP. The dependence of the maximum of the current transients on concentration of ADP was reproduced by mathematical simulations. They fit the data well on the assumption that the rate-limiting reaction step of the Na(+)-translocation partial reaction is the conformational transition E1/E2.
authors.affiliation: Department of Biology, University of Konstanz, Germany.
authors.forename: H J
authors.initials: HJ
authors.lastname: Apell
authors.forename: A
authors.initials: A
authors.lastname: Schneeberger
authors.forename: V S
authors.initials: VS
authors.lastname: Sokolov
mesh_terms: D000255:Adenosine Triphosphate / Q000502:physiology; D001692:Biological Transport / Q000502:physiology; D004563:Electrochemistry; D004798:Enzymes / Q000378:metabolism; D007700:Kinetics; D010766:Phosphorylation; D011487:Protein Conformation; D012964:Sodium / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry / Q000378:metabolism*
chemical_list: D004798:Enzymes; D000255:Adenosine Triphosphate; D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase