congresos y reuniones científicas
Unexpected effects of K+ and ATP on the thermal stability of Na+,K+-ATPase
Congreso; Latin American Crosstalk In Biophysics And Physiology; 2015
Institución organizadora:
Seccional Biofísica de la Sociedad Uruguaya de Biociencias (, la Sociedad Argentina de Biofísica y LAFeBS
Na⁺,K⁺ -ATPase is an integral membrane protein which couples ATP hydrolysis to the transport of three Na⁺ out and two K⁺ into the cell. During this catalytic cycle, the enzyme interconvert between two conformers, E1 and E2. 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]. The aim of this work is to 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 (which reflects structural changes in the ATP binding domain).We observed that thermal inactivation in all conditions tested followed a first-order kinetic. The decrease of ATPase activity is concomitant with the conformational change detected by Trp fluorescence. A clear stabilization effect was observed for all three ligands, due to both enthalpic and entropic contributions. 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.Competition experiments between ATP and K+ showed that at constant [ATP] the rate inactivation coefficient presented a biphasic dependence on [K+], which progressively shifts to the right as [ATP] increased. In the presence of nucleotide, destabilization of the enzyme was observed at low [K+], while stabilization occurred as the cation concentration increased. This complex behavior is not what expected for a simple competition between the enzyme and two ligands that individually protect the enzyme against thermal inactivation. A model that includes enzyme species with none, one or two K⁺ and simultaneously one molecule of ATP bound could explain the experimental data. This would be possible if ligand binding stabilizes the enzyme except for the specie with one K⁺ and one ATP bound. [1]Kaufman SB, González-Flecha FL, González-Lebrero RM. Opposing effects of Na+ and K+ on the thermal stability of Na⁺,K⁺-ATPase. J Phys Chem B. 116(10), 3421-9 (2012).