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

MARIELA S. FERREIRA-GOMES; MÓNICA R. MONTES; SANTIAGO E. FARAJ; JUAN PABLO F.C. ROSSI; MERCEDES CENTENO; ROLANDO C. ROSSI

Otsu, Siga

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

Hiroshi Suzuki (Asahikawa Medical University), Haruo Ogawa (University of Tokyo), Chikashi Toyoshima (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 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 uM, 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 uM eosin Y in a medium containing 1 mM KCl and different concentrations of NaCl and EGCg. The K 0.5 for Na + for the increment in eosin fluorescence in the absence of EGCg was 2.2 mM; this value decreased to 0.9, 0.6 and 0.1 mM in the presence of 100, 200 and 500 uM EGCg, respectively. Results from experiments measuring tighly bound 22 Na + to the Na,K-ATPase in the presence of 100 uM EGCg show that this increment in affinity for Na + is compatible with the formation of a state containing three occluded Na+.