Interaction of ATP, Calcium and Vanadate with the Plasma Membrane Calcium pump: The regulatory effect of ATP

IC MANGIALAVORI; M. FERREIRA GOMES; RODOLFO M GONZÁLEZ LEBRERO; RC. ROSSI; JPFC ROSSI

Congreso; XLI Reunión anual de la Sociedad Argentina de Biofísica; 2012

Sociedad Argentina de Biofísica

P { margin-bottom: 0.21cm; }A:link { } The Plasma Membrane Calcium ATPase (PMCA) is a calmodulin-regulated P-type ATPase responsible for the maintenance of low intracellular concentration of Ca2+ in most eukaryotic cells. The current kinetic model proposes that the enzyme exists in two main conformations, E1 and E2. After binding of intracellular Ca2+ to high-affinity sites, E1 can be phosphorylated by ATP with formation of the intermediate E1P, which results in occlusion of bound Ca2+ 1. After a conformational transition to E2P, Ca2+ would be released to the extracellular medium from low-affinity sites, followed by the hydrolysis of the phosphoenzyme to E2 and a new conformational transition to E1. The aim of this work was to study the PMCA calcium transport mechanism through the characterization of calcium occlusion/deocclusion partial reactions. We used PMCA obtained by Baculovirus–mediated expression in Sf9 insect cells and isolated as microsomal membranes. Calciumocclusion was measured using the method described by Ferreira-Gomes et al. (2011)1. We performed calciumocclusion measurements as a function of time in the presence of ATP, and found no significant differences among the rate coefficients for the different Ca2+ concentrations tested. This result indicates that steady-state calcium occlusion is a very high-affinity phenomenon. Furthermore, we measured the time course of calcium deocclusion, which showed a biphasic behavior. The fast phase was compatible with the ATPase activity of the pump under the same experimental conditions. When the calcium deocclusion was carried out in the presence of 500 mM Ca2+ the velocity of fast phase was lower than in the absence of the cation. This suggests that the Ca2+ may compete with Mg2+ 2 which regulates the phosphorylation/dephosphorylation steps in the PMCA reaction cycle. Ferreira-Gomes, M. S., González-Lebrero, R. M., de la Fuente, M. C., Strehler, E. E., Rossi, R. C., Rossi, J. P. (2011) J Biol Chem. 286, 32018-32025 Penniston J. T. (1982) Biochim Biophys Acta.  688, 735-739 With grants of UBACYT, CONICET and ANCYT.