Structural significance of the plasma membrane calcium pump oligomerization.
The oligomerization of the plasma membrane calcium pump (PMCA) in phospholipid/detergent micelles was evaluated using a combined spectroscopic and kinetic approach and related to the enzyme stability. Energy transfer between fluorescein-5'-isothiocyanate and eosin-5'-isothiocyanate attached to different PMCA molecules was used to determine the dissociation constant of dimeric PMCA (140 +/- 50 nM at 25 degrees C) and characterize the time course of dimerization. The enzyme thermal 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 treatment leads to the formation of inactive monomers that associate only with native monomers. These mixed dimers are formed with a kinetic coefficient that is half that determined for the native dimers. We proposed a model for PMCA thermal inactivation that considers the equilibria among dimers, monomers, and mixed dimers, and the inactivation of the last two species through irreversible steps. The numerical resolution of the differential equations describing this model fitted to the experimental data allowed the determination of the model coefficients. This analysis shows that thermal inactivation occurs through the denaturation of the monomer, which lifetime is 25 min at 44 degrees C. The obtained results suggest that PMCA dimerization constitutes a mechanism of self protection against spontaneous denaturation.
Levi
Valeria
V
Departamento de Química Biológica, Instituto de Quimica y Fisicoquimica Biologicas, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, 1113 Buenos Aires, Argentina.
Rossi
Juan P F C
JP
Castello
Pablo R
PR
González Flecha
F Luis
FL
D000252:Calcium-Transporting ATPases / Q000097:blood / Q000737:chemistry* / Q000378:metabolism; D003902:Detergents; D019281:Dimerization; D004795:Enzyme Stability; D004910:Erythrocyte Membrane / Q000201:enzymology*; D006801:Humans; D007700:Kinetics; D046911:Macromolecular Substances; D008823:Micelles; D010743:Phospholipids; D013816:Thermodynamics
D003902:Detergents; D046911:Macromolecular Substances; D008823:Micelles; D010743:Phospholipids; D000252:Calcium-Transporting ATPases