Epigallocatechin-3-gallate promotes tight binding of Na+ to the Na,K-ATPase

FARAJ, S.E.; FERREIRA-GOMES, M S. ; SAINT MARTIN, E.M.; CENTENO, M. M.; ROSSI, J.P.F.C.; MONTES, M. R.; ROSSI, R. C.

Congreso; The 15th International Conference on Na, K-ATPase and Related Transport ATPases; 2017

Asahikawa Medical University, University of Tokyo

One of the less studied aspects of the Na,K-ATPase reaction cycle is the kinetics of formation and breakdown of the intermediates involved in the transport of Na+. According to the Albers-Post model, binding of 3 intracellular Na+ to the enzyme triggers phosphorylation by ATP in the presence of Mg2+ and Na+ becomes occluded in the phosphorylated intermediate E1P. With the only exception of measurements in equilibrium by Matsui and Homareda (1982), occlusion of Na+ has been reported in inhibited enzyme, in the presence of oligomycin or Cr-ATP, and in partially proteolyzed enzyme. The aim of the present work is to develop a procedure for measuring the kinetics of Na+ occlusion in the Na,K-ATPase during the normal functioning of the reaction cycle. For this, states with occluded Na+ need to be rapidly stabilized and isolated. As a stabilizing reagent, we propose using epigallocatechin-3-gallate (EGCg), which inhibits Na,K-ATPase activity with high affinity (less than 1 micromolar) (Ochiai et al., 2009).In this work, we show that EGCg increases the affinity of Na,K-ATPase for Na+ by stabilizing a state containing occluded Na+. Experiments were carried out at 25 °C in media with imidazole-HCl 25 mM, pH 7.4, using Na,K-ATPase partially purified from pig kidney. Enzyme was incubated with 0.4 micromolar eosin Y in a medium containing 1 mM KCl and different concentrations of NaCl and EGCg. The K0.5 for Na+ for the increment in eosin fluorescence in the absence of EGCg was 2.2 mM, which decreased to 0.9, 0.6 and 0.1 mM in the presence of 100, 200 and 500 micromolar EGCg, respectively. Results from experiments measuring tightly bound 22Na+ to the Na,K-ATPase in the presence of 100 micromolar EGCg show that this increment in affinity for Na+ is compatible with the formation of a state containing three occluded Na+.References1. Matsui, H and Homareda, H. J Biochem. 92, 193-217, 19822. Ochiai, H et al. Biochem Pharmacol. 78, 1069-1074, 2009With grants from Agencia Nacional de Promoción Científica y Tecnológica (PICT 2012 - 2014 1053), Consejo Nacional de Investigaciones Científicas y Técnicas (PIP 11220150100250CO), and Universidad de Buenos Aires (Ciencia y Técnica, grant 2014-2017 20020130100302BA), Argentina.