Epigallocatechin-3-gallate increases the affinity for Na+ in the Na,K-ATPase

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

Congreso; Reunión Conjunta de Sociedades de Biociencias; 2017

The kinetics of formation and breakdown of the intermediates involved in the transport of Na+ is one of the less studied aspects of the Na,K-ATPase reaction cycle. According to the Albers-Post model, binding of 3 intracellular Na+ to the E1 state of the enzyme triggers phosphorylation by ATP in the presence of Mg2+ and Na+ becomes occluded in the phosphorylated intermediate E1P. Na+ is released to the extracellular medium after the E1PE2P conformational transition. Occlusion of Na+ has only 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.In this work, we propose to use epigallocatechin-3-gallate (EGCg) as a stabilizing agent (Ochiai et al., 2009, Biochem. Pharmacol., 78:1069-1074) and a rapid-filtration procedure to isolate the species with tightly bound 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. To evaluate the effects of EGCg on the affinity for Na+, enzyme was incubated with eosin Y in a medium containing RbCl and different concentrations of NaCl and EGCg. The K0.5 for Na+ for the increment in eosin fluorescence decreased as [EGCg] increased. Measurements of 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 stabilization of a state containing occluded Na+. Addition of 100 micromolar EGCg to the washing solution is sufficient to ?instantaneously freeze? the reactions of formation and breakdown of Na+-bound states during the normal functioning of the reaction cycle.Our results show that EGCg is a good stabilizing agent for characterizing the steps involved in the transport of Na+.Keywords: Na,K ATPase, cation transport, Na+ occlusion, epigallocatechin 3 gallateMalén Saint-Martin, Santiago E. Faraj, Mariela S. Ferreira-Gomes, Mercedes Centeno, Juan Pablo F.C. Rossi, Mónica R. Montes and Rolando C. RossiIQUIFIB, Facultad de Farmacia y Bioquímica, Universidad de Buenos AiresJunín 956, Buenos Aires, C1113AAD, ArgentinaWith grants from Consejo Nacional de Investigaciones Científicas y Técnicas, Universidad de Buenos Aires, and Agencia Nacional de Promoción Científica y Tecnológica, Argentina.