Opposing Effects of Na+ and K+ on the Thermal Stability of Na+,K+-ATPase B

Folding and structural stability are key factors for the proper biological function of proteins. Na⁺,K⁺-ATPase is an integral membrane protein involved in the active transport of Na⁺ and K⁺ across the plasma membrane. In this work we characterized the effects of K⁺ and Na⁺ on the thermal inactivation of Na⁺,K⁺-ATPase, evaluating both catalytic and transport capacities of the pump. Both activities of the enzyme decrease with the preincubation time as first-order kinetics. The thermal inactivation of Na⁺,K⁺-ATPase is simultaneous with a conformational change detected by tryptophan and 1-aniline-8-naphtalenesulfonate (ANS) fluorescence. The kinetic coefficient of thermal inactivation was affected by the presence of Na⁺ and K⁺ (or Rb⁺) and the temperature of the preincuabtion media. Our results show that K⁺ or Rb⁺ stabilize the enzyme, while Na⁺ decreases the stability of Na⁺,K⁺-ATPase. Both effects are exerted by the specific binding of these cations to the pump. Also, we provided strong evidence that the Rb⁺ (or K⁺) stabilization effect is due to the occlusion of these cations into the enzyme. Here, we proposed a minimal kinetic model that explains the behavior observed in the experimental results and allows a better understanding of the results presented by other researchers. The thermal inactivation process was also analyzed according to Kramer’s theory.