Thermal inactivation of the NA+,K+-ATPase, ATP and Mg2+ effects

MA. PLACENTI; SB. KAUFMAN; FL. GONZÁLEZ FLECHA; RM. GONZÁLEZ LEBRERO

Carlos Paz

Congreso; XLII reunión anual de la Sociedad Argentina de Biofísica; 2013

Sociedad Argentina de Biofísica

Folding and stability are key factors for the proper biological function of proteins, therefore the importance of its study. 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 .The aim of this work is to characterize the effect of natural ligands (ATP and Mg2+)on the thermal inactivation of Na+,K+-ATPase. In a typical thermal inactivation experiment, the enzyme is incubated for different time periods at several temperatures in the presence or absence of the ligand. The functional or structural integrity of the protein is followed by measurement of some property, in our case, ATPase activity, Trp fluorescence and circular dichroism spectroscopy. 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. Additionally, circular dichroism experiments showed a slight change in the secondary structure when the ATPase was fully inactivated. A clear stabilization of the enzyme was observed when ATP or Mg2+ was present in the incubation media. Even though Na or ATP are known to displacetheequilibrium of the enzyme towards E1 conformer, our results showed that those ligands have opposite effects in terms of thermal stability of the Na+,K+-ATPase. With grants from: UBACyT, CONICET and ANPCyT.