“Thermal Stability of the Plasma Membrane Calcium Pump. Quantitative Analysis of its Dependence on Lipid-Protein Interactions”.

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

JOURNAL OF MEMBRANE BIOLOGY

Springer Verlag

Lugar: New York; Año: 2000 p. 215 - 225

0022-2631

Abstract. Thermal stability of plasma membrane Ca2+pump was systematically studied in three micellar systemsof different composition, and related with the interactionsamphiphile-protein measured by fluorescenceresonance energy transfer. Thermal denaturation wascharacterized as an irreversible process that is well describedby a first order kinetic with an activation energyof 222 ± 12 kJ/mol in the range 33–45°C. Upon increasingthe mole fraction of phospholipid in the mixed micelleswhere the Ca2+ pump was reconstituted, the kineticcoefficient for the inactivation process diminished until itreached a constant value, different for each phospholipidspecies. We propose a model in which thermal stabilityof the pump depends on the composition of the amphiphilemonolayer directly in contact with the transmembraneprotein surface. Application of this model showsthat the maximal pump stability is attained when 80% ofthis surface is covered by phospholipids. This analysisprovides an indirect measure of the relative affinity phospholipid/detergent for the hydrophobic transmembranesurface of the protein (KLD) showing that those phospholipidswith higher affinity provide greater stability to theCa2+ pump. We developed a method for directly measureKLD by using fluorescence resonance energy transferfrom the membrane protein tryptophan residues to apyrene-labeled phospholipid. KLD values obtained bythis procedure agree with those obtained from the model,providing a strong evidence to support its validity.