Partial Reactions of the Na,K-ATPase: Determination of Activation Energies and an Approach to Mechanism
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Partial Reactions of the Na,K‑ATPase: Determination of Activation Energies and an Approach to Mechanism Hans‑Jürgen Apell1 · Milena Roudna1 Received: 20 October 2020 / Accepted: 5 November 2020 © The Author(s) 2020
Abstract Kinetic experiments were performed with preparations of kidney Na,K-ATPase in isolated membrane fragments or reconstituted in vesicles to obtain information of the activation energies under turnover conditions and for selected partial reactions of the Post-Albers pump cycle. The ion transport activities were detected with potential or conformation sensitive fluorescent dyes in steady-state or time-resolved experiments. The activation energies were derived from Arrhenius plots of measurements in the temperature range between 5 °C and 37 °C. The results were used to elaborate indications of the respective underlying rate-limiting reaction steps and allowed conclusions to be drawn about possible molecular reaction mechanisms. The observed consequent alteration between ligand-induced reaction and conformational relaxation steps when the Na,KATPase performs the pump cycle, together with constraints set by thermodynamic principles, provided restrictions which have to be met when mechanistic models are proposed. A model meeting such requirements is presented for discussion. Graphic Abstract
Keywords Sodium pump · Active ion transport · Post-Albers cycle · Reaction kinetics · Fluorescence · Molecular mechanism
Introduction In most animal cells electrochemical potential gradients across the cytoplasmic membrane are generated directly or indirectly by the Na,K-ATPase. From a thermodynamical point of view, this protein can be understood as a molecular * Hans‑Jürgen Apell h‑j.apell@uni‑konstanz.de 1
Department of Biology, University of Konstanz, 78464 Konstanz, Germany
machine that converts chemical energy, stored in the compound ATP, to an electrochemical potential gradient across the cell membrane by pumping three sodium ions out of the cytoplasm and two potassium ions in opposite direction per each turnover. Numerous publications and reviews are available presenting information on structural aspects (Morth et al. 2007; Shinoda et al. 2009; Kanai et al. 2013; Nyblom et al. 2013), enzymatic and transport properties(Glynn 1985; Kaplan 1985; Jørgensen et al. 2003; Apell 2019), electrogenicity (De Weer et al. 1988; Apell 2004; Gadsby 2009), and energetics (Läuger 1984, 1991).
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In its normal mode of operation, the enzyme performs a pump cycle composed of conformational transitions as well as ligand binding and release steps. Spectroscopic and proteolytic studies indicated that the enzyme can assume two principal conformations, designated as E 1 and E 2. The E1 conformation is stabilized by Na+ and characterized by ion-binding sites facing the cytoplasm; the E2 conformation is stabilized by K+ and the ion-binding sites are accessible from the extracellular medium in the phosphorylated state of E2. From enzymatic and transport studies under various conditions, a rea
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