PLASMA MEMBRANE CALCIUM PUMP ACTIVITY MODULATION BY MICELLAR SYSTEM COMPOSITION

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

Búzios

Congreso; VII IBEROAMERICAN CONGRESS OF BIOPHYSICS; 2009

Introduction: Plasma membrane composition influences activity of membrane proteins by differential interaction of its components with proteins. The complexity of natural membranes complicates the analysis of these interactions in situ, so these studies are commonly carried out in artificial amphiphile systems, e.g. mixed detergent-lipid micelles. Objective: Study how micellar system composition affects membrane protein activity using the Plasma Membrane Calcium Pump (PMCA) as a model. Materials: Purified PMCA was incubated in buffer with different amounts of lipids and detergent (C12E10). ATPase activity was measured as the initial rate of Pi release from ATP. Results: C12E10 solubilized PMCA shows no activity, but addition of phospholipids restores activity in a concentration dependent way. Also, addition of C12E10 to phospholipid supplemented PMCA leads to inactivation at a level depending on the amount of phospholipids used to reconstitute the enzyme. These results converge if we represent the ATPase activity as a function of the mole fraction of phospholipids in the micelle (Xpl). Soybean phospholipids gave three times more maximal activity than  PC/PE mixtures. 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 Xpl with ATPase activity. The model includes a parameter KLD that accounts for the exchange between amphiphiles, allowing to estimate the relative affinity of each phospholipid for the transmembrane region. Our experiments showed that phospholipids producing higher activation also had higher KLD values, and these values were similar to those determined by fluorescence methods. Conclusion: These results provide evidence that membrane protein activity is modulated by the composition of the amphiphile monolayer directly in contact with the transmembrane protein surface.