EPIGALLOCATECHIN-3-GALLATE INCREASES THE AFFINITY FOR NA+ IN THE NA,KATPASE

ELINA MALEN SAINT MARTIN; SANTIAGO FARAJ; MARIELA FERREIRA GOMES; MERCEDES CENTENO; JUAN PABLO ROSSI; MÓNICA MONTES; ROLANDO C.ROSSI

Congreso; Reunión Conjunta Sociedades de Biociencia; 2017

Sociedades de Biociencia

The kinetics of formation and breakdown of the intermediates involvedin the transport of Na+ is one of the less studied aspectsof the Na,K-ATPase reaction cycle. According to the Albers-Postmodel, binding of 3 intracellular Na+ to the E1 state of the enzymetriggers phosphorylation by ATP in the presence of Mg2+ and Na+becomes occluded in the phosphorylated intermediate E1P. Na+ isreleased to the extracellular medium after the E1P®E2P conformationaltransition. Occlusion of Na+ has only been reported in inhibitedenzyme, in the presence of oligomycin or Cr-ATP, and in partiallyproteolized enzyme.The aim of the present work is to develop a procedure for measuringthe kinetics of Na+ occlusion in the Na,K-ATPase during the normalfunctioning of the reaction cycle. For this, states with occludedNa+ 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 specieswith tightly bound Na+.Experiments were carried out at 25 °C in media with imidazole-HCl25 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 wasincubated with eosin Y in a medium containing RbCl and differentconcentrations of NaCl and EGCg. The K0.5 for Na+ for the incrementin eosin fluorescence decreased as [EGCg] increased. Measurementsof tightly bound 22Na+ to the Na,K-ATPase in the presenceof 100 mM EGCg show that this increment in affinity for Na+ iscompatible with the stabilization of a state containing occluded Na+.Addition of 100 mM EGCg to the washing solution is sufficient to ?instantaneouslyfreeze? the reactions of formation and breakdown ofNa+-bound states during the normal functioning of the reaction cycle.Our results show that EGCg is a good stabilizing agent for characterizingthe steps involved in the transport of Na+.