Calcium occlusion in the plasma membrane Ca2+-ATPase

M. FERREIRA GOMES; RODOLFO M GONZÁLEZ LEBRERO; MC DE LA FUENTE; EE. STREHLER; RC. ROSSI; JPFC ROSSI

JOURNAL OF BIOLOGICAL CHEMISTRY

AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC

Año: 2011 vol. 286 p. 32018 - 32025

0021-9258

In this work, we set out to identify and characterize the calcium occluded intermediate(s) of the plasma membrane Ca2+-ATPase (PMCA) to study the mechanism of calcium transport.To this end, we developed a procedure for measuring the occlusion of Ca2+ in microsomes containing PMCA. This involves a system for overexpression of the PMCA and the use of a rapidmixing device combined with a filtration chamber, allowing the isolation of the enzyme and quantification of retained calcium. Measurements of retained calcium as a function of the Ca2+concentration in steady state showed a hyperbolic dependence with an apparent dissociation constant of 12 +- 2.2 uM, which agrees with the value found through measurements of PMCA activity in the absence of calmodulin. When enzyme phosphorylation and the retained calcium were studied as a function of time in the presence of LaIII (inducing accumulation of phosphoenzyme in the E1P state), we obtained apparent rate constants not significantly different from each other. Quantification of EP and retained calcium in steady state yield a stoichiometry of one mole of occluded calcium per mole of phosphoenzyme. These results demonstrate for the first time that one calcium ion becomes occluded in the E1P-phosphorylated intermediate of the PMCA