Stabilizing effects of ATP, Mg2+ and K+ on the NA+ ,K+ -ATPase thermal inactivation

PLACENTI, M.A., KAUFMAN, S.B. GONZÁLEZ FLECHA, F.L. AND GONZÁLEZ-LEBRERO, R.M.

Sierra de la Ventana, Buenos Aires

Congreso; XLIII Reunion anual de la Sociedad Argentina de Biofisica; 2014

Sociedad Argentina de Biofisica

Na+,K+ -ATPase is an integral membrane protein which couplesATP hydrolysis to the transport of three Na+ out and two K into the cell, cycling between the E1 and E2 conformers. In a previous work we show that Na+ and K+, which leads the enzyme to E1 and E2 respectively, presented opposite effects on thermal stability of the pump [1]. In this work, we characterize the effect of some natural ligands on the protein thermal stability. Thermal inactivation was performed incubating the enzyme in the presence or absence of ATP, Mg2+ or K+ for different time periods and temperatures. After this incubation we measured ATPase activity, Trp fluorescence and Eosin Y binding. It is known that this fluorescent probe binds to the ATP binding site, and therefore reflects the structural changes in it. Ours results showed that in all conditions tested, ATPase activity decreased following a first-order kinetic, concomitant with the change in both Trp and eosin fluorescence. Transition state theory and thermodynamic activation parameters (∆G?, ∆H? y ∆S?) were used to analyze and interpret the data. A clear stabilization effect was observed for all three ligands, due to both enthalpic and entropic contributions. This effect is more important for K+, which displaces the equilibrium of the enzyme towards E2. Even though ATP is known to displace the equilibrium to the E1 as Na+, these two ligands have opposite effects in terms of thermal stability of the Na ,K -ATPase.