Evidence of direct binding of G-actin to PMCA by Surface Plasmon Resonance

DALGHI M, FERNÁNDEZ M, MALCHIODI E, ROSSI JP.

Salta

Congreso; SAB 2010 Workshop CeBEM-Protein Society Meeting; 2010

SAB-Protein Society

Evidence of direct binding of G-actin to PMCA by Surface Plasmon Resonance.   Marianela G. Dalghia, Marisa M. Fernándezb, Emilio L. Malchiodib and Juan Pablo F.C. Rossia.   aIQUIFIB-Departamento de Química Biológica, Facultad de Farmacia y Bioquímica, UBA. Junín 956, 1113, Buenos Aires, Argentina  bIDEHU-Cátedra de Inmunología, Facultad de Farmacia y Bioquímica, UBA. Junín 956, 1113, Buenos Aires, Argentina.   mdalghi@qb.ffyb.uba.ar   Previous studies of our laboratory suggest that the main isoform of the plasma membrane calcium ATPase (PMCA) expressed in erythrocytes, PMCAh4b, associates with the actin cytoskeleton and its catalytic activity can be regulated through this interaction. Both states of actin (G and F actin) are able to interact with PMCA but they show different behavior. Apparently, G-actin and short oligomers enhance the catalytic activity of the pump, while F-actin seems to inhibit it. To further explore the interaction between PMCA and G-actin, we performed binding experiments based on Surface Plasmon Resonance technology (SPR) using purified PMCAh4b from human red blood cells and purified rabbit muscle G-actin on a BIAcore T100 system. Our strategy consisted in immobilizing G-actin to the sensor chip surface while PMCA constituted the analyte (range of concentrations from 62.5 nM to 2 µM). The sensograms obtained showed an increase in the response (RU) as a function of PMCA concentration with a saturable binding. The unspecific binding values were subtracted from the response obtained in the control flow cell (without immobilized G-actin). Using this experimental approach it was possible to confirm a direct binding interaction and to determine in a preliminary way the apparent KD (1 µM). These results revealed a specific and high affinity direct binding interaction between PMCA and G-actin. With grants of ANPCYT, CONICET and UBA.