Calcium Occlusion in the Phosphorylated Intermediate of Plasma Membrane Ca2+-ATPase

FERREIRA GOMES MS; GONZÁLEZ-LEBRERO RM; DE LA FUENTE MC; ROSSI RC; ROSSI JPFC

Salta

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

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 would result in occlusion of bound Ca2+. After a conformational transition to E2P, Ca2+ would be released to the extracellular medium from low-affinity sites, the phosphoenzyme is hydrolyzed and the resulting E2 intermediate state undergoes a new conformational transition to E1. The aim of this work was to identify the calcium occlusion intermediate and to assess whether the occlusion of calcium is concomitant with the phosphorylation of the enzyme by ATP.                 We used PMCA obtained by Baculovirus–mediated expression in Sf9 insect cells and isolated as microsomal membranes. Ca2+ occlusion was measured using the method described by Rossi et al. (1999) with minor modifications. We measured the amount of the phosphorylated intermediate and of calcium occluded in the PMCA as a function of time, under the same experimental conditions, and obtained similar apparent rate constants. Quantification of EP and occluded calcium in steady state yield a stoichiometry of one mole of occluded calcium per mole of phosphoenzyme. Therefore, we can conclude that formation of phosphoenzyme and calcium occlusion are simultaneous events.