EFFECTS OF 1,2-DIOLEOYL-SN-GLYCERO-3-PHOSPHOCHOLINE ON THE ACTIVITY OF PLASMA MEMBRANE CALCIUM PUMP

MARTÍN DODES TRAIAN; DIEGO IGNACIO CATTONI; VALERIA LEVI; FRANCISCO LUIS GONZÁLEZ FLECHA

Los Cocos, Córdoba

Congreso; XXXVIII ANUAL MEETING OF THE BIOPHYSICAL SOCIETY OF ARGENTINA; 2009

Sociedad Argentina de Biofísica

Membrane protein activity can be modulated by differential interaction of membrane components. The analysis of these interactions is commonly carried out in artificial amphiphile systems, like membrane proteins reconstituted in lipid vesicles or mixed detergent-lipid micelles.  In this work, the effect of lipid protein interaction on membrane protein activity was studied using the Plasma Membrane Calcium Pump (PMCA) in a mixed micellar system composed of the detergent C12E10 and 1,2-Dioleoyl-sn-Glycero-3-phosphocholine (DOPC) as a model.  PMCA is a calcium transport P-ATPase present in the plasma membrane of all eukaryotic cells constituted of a single 134 kDa polypeptidic chain that spans the membrane 10 times.PMCA was purified by affinity chromatography in a calmoduline-agarose column obtaining the protein reconstituted in C12E10 micelles.  This protein preparation showed no enzymatic activity.  The addition of increasing amounts of DOPC to that detergent-protein system produces a reversible increase in activity reaching a maximum value. In order to study if the transduction between micellar composition and activity could be linked to changes in secondary or tertiary structure, we analyzed the far UV Circular Dichroism (CD) and tryptophan fluorescence spectra in the DOPC/C12E10 range used for the activity assays.  No changes were detected indicating that no major structural changes occur during the activation process.   The relative affinity between DOPC and C12E10 for the PMCA transmembrane region was evaluated by FRET between PMCA tryptophan residues and a fluorescent-labeled PC [1].  To account for these results, we proposed a model, in which PMCA activity depends on the composition of the amphiphile monolayer covering the transmembrane protein surface. Our model shows good agreement with experimental data, linking amphiphile/protein interactions with ATPase activity.