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

FERREIRA GOMES, MARIELA; MONTES, MÓNICA R.; CENTENO, MERCEDES; ROSSI ROLANDO; FARAJ, SANTIAGO; ROSSI JUAN PABLO

Otsu City, Shiga

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

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 proteolized 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. Stabilization can be achieved, in principle, with a reagent like oligomycin. However, the low solubility of oligomycin prevents reaching concentrations sufficiently high as to allow a quick combination with the enzyme. An alternative is to use epigallocatechin-3-gallate (EGCg), which inhibits Na,K-ATPase activity with high affinity (less than 1 M, 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 M 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 2.4 mM; this value decreased to 1.6, 1.1 and 0.2 mM in the presence of 100, 200 and 500 M EGCg, respectively. Results from experiments measuring tighly bound 22Na+ to the Na,K-ATPase in the presence of 100 M 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 Consejo Nacional de Investigaciones Científicas y Técnicas, Universidad de Buenos Aires, and Agencia de Promoción Científica y Tecnológica, Argentina.