"Structural significance of the plasma membrane calcium pump oligomerization"

LEVI, V; ROSSI, J.P.F.C.; CASTELLO PR; GONZÁLEZ FLECHA, F.L.

BIOPHYSICAL JOURNAL

Biophysical Society

Lugar: New York; Año: 2002 vol. 82 p. 437 - 446

0006-3495

ABSTRACT The oligomerization of the plasma membrane calcium pump (PMCA) in phospholipid/detergent micelles wasevaluated using a combined spectroscopic and kinetic approach and related to the enzyme stability. Energy transfer betweenfluorescein-5-isothiocyanate and eosin-5-isothiocyanate attached to different PMCA molecules was used to determine thedissociation constant of dimeric PMCA (140 50 nM at 25°C) and characterize the time course of dimerization. The enzymethermal stability at different dimer/monomer ratios was evaluated, quantifying the kinetic coefficient of thermal inactivation.This coefficient decreases with PMCA concentration, becoming approximately constant beyond 300 nM. Thermal treatmentleads to the formation of inactive monomers that associate only with native monomers. These mixed dimers are formed witha kinetic coefficient that is half that determined for the native dimers. We proposed a model for PMCA thermal inactivationthat considers the equilibria among dimers, monomers, and mixed dimers, and the inactivation of the last two species throughirreversible steps. The numerical resolution of the differential equations describing this model fitted to the experimental dataallowed the determination of the model coefficients. This analysis shows that thermal inactivation occurs through thedenaturation of the monomer, which lifetime is 25 min at 44°C. The obtained results suggest that PMCA dimerizationconstitutes a mechanism of self protection against spontaneous denaturation.