IQUIFIB   02644
INSTITUTO DE QUIMICA Y FISICOQUIMICA BIOLOGICAS "PROF. ALEJANDRO C. PALADINI"
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
Thermal stability of the Na+/K+ ATPase, effect of ligands
Autor/es:
SB. KAUFMAN; FL. GONZÁLEZ FLECHA; RM. GONZÁLEZ-LEBRERO
Lugar:
University of Aarhus, Aarhus, Denmark.
Reunión:
Conferencia; 12th International ATPase Conference. Na,K-ATPase and Related Transport ATPases of P-type: Structures, Mechanisms, and Roles in Health and Disease; 2008
Resumen:
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