Unexpected effects of K+and ATP on the thermal stability of Na+,K+-ATPase

M. AGUEDA PLACENTI; SERGIO B. KAUFMAN; F. LUIS GONZÁLEZ FLECHA; RODOLFO M. GONZÁLEZ LEBRERO

Concordia

Congreso; Latin American Crosstalk In Biophysics And Physiology; 2015

Sociedad Argentina de Biofísica

Na+,K+ -ATPase is an integral membrane protein which couples ATPhydrolysis to the transport of three Na+ out and two K+ into the cell.During this catalytic cycle, the enzyme interconvert between twoconformers, E 1 and E 2 . In a previous work we show that Na+ and K+,which leads the enzyme to E 1 and E 2 respectively, presented oppositeeffects on thermal stability of the pump [1]. The aim of this work is tocharacterize the effect of some natural ligands on the protein thermalstability. Thermal inactivation was performed incubating the enzymein the presence or absence of ATP, Mg2+ or K+ for different timeperiods and temperatures. After this incubation we measured ATPaseactivity, Trp fluorescence. We observed that thermal inactivation in allconditions tested followed a first-order kinetic. The decrease ofATPase activity is concomitant with the conformational changedetected by Trp fluorescence. A clear stabilization effect was observedfor all three ligands, due to both enthalpic and entropic contributions.Even though ATP is known to displace the equilibrium to the E1 asNa+, these two ligands have opposite effects in terms of thermalstability of the Na+ ,K+ -ATPase.Competition experiments between ATP and K+ showed that atconstant [ATP] the rate inactivation coefficient presented a biphasicdependence on [K+], which progressively shifts to the right as [ATP]increased. In the presence of nucleotide, destabilization of theenzyme was observed at low [K+], while stabilization occurred as thecation concentration increased. This complex behavior is not whatexpected for a simple competition between the enzyme and twoligands that individually protect the enzyme against thermalinactivation. A model that includes enzyme species with none, one ortwo K+ and simultaneously one molecule of ATP bound could explainthe experimental data. This would be possible if ligand bindingstabilizes the enzyme except for the specie with one K+ and one ATPbound.