Thermal stability of the Na+/K+ ATPase, effect of ligands

SB. KAUFMAN; FL. GONZÁLEZ FLECHA; RM. GONZÁLEZ-LEBRERO

University of Aarhus, Aarhus, Denmark.

Conferencia; 12th International ATPase Conference. Na,K-ATPase and Related Transport ATPases of P-type: Structures, Mechanisms, and Roles in Health and Disease; 2008

<!-- /* Style Definitions */ p.MsoNormal, li.MsoNormal, div.MsoNormal {mso-style-parent:""; margin:0cm; margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:12.0pt; font-family:"Times New Roman"; mso-fareast-font-family:"Times New Roman";} @page Section1 {size:612.0pt 792.0pt; margin:70.85pt 3.0cm 70.85pt 3.0cm; mso-header-margin:36.0pt; mso-footer-margin:36.0pt; mso-paper-source:0;} div.Section1 {page:Section1;} --> Folding and stability are an important factors to understand protein biological functions. However, little is known about the mechanisms stabilizing the membrane proteins. In this work the thermal stability of the Na+/K+-ATPase from pig kidney was studied using a kinetic approach. Also, the effect of different ligands to protect the enzyme from inactivation was analyzed. We have measured the ATPase activity and the amount of occluded Rb+ (via the direct route) after preincubation of the enzyme at several temperatures during different time periods. The Na+/K+-ATPase activity decline irreversibly with the incubation time along a single exponential, suggesting a two-state process involving fully-active and inactive molecules. We observed that both Na+/K+-ATPase activity and Rb+ occlusion decrease with non significant differences on their inactivation rate coefficients when the enzyme was preincubated in media with the same composition and temperature. We also tested the different velocities of inactivation in media containing either Na+, where the pump is in E1 conformation, or K+ (or its congener Rb+) where it is in E2 conformation. The values of inactivation coefficients, in medium with Na+ or in absence of cation, noticeably increased when the incubation temperature increased beyond 51ºC. Nevertheless, this temperature was 55ºC when the incubation media contained 40 mM K+ or Rb+. A further characterization of the inactivation process was studied using the Arrhenius plot of the inactivation rate coefficients equation. The values of activation energy (Ea) obtained without added cations or with Na+ in the preincubation media (Ea = 444 ± 38 and 428 ± 37 KJ/mol, respectively) were significantly lower than those obtained in the presence of K+ or Rb+ (Ea = 603 ± 69 and 664 ± 46 KJ/mol, respectively). These values of Ea indicate that the presence of K+ or Rb+ in the incubation media produce stabilization of the enzyme, possibly because the enzyme is in E2 conformation. With grants from ANPCyT, CONICET and UBA