Calcium Occlusion of the Plasma membrane ATPase

MARIELA S. FERREIRA-GOMES; RODOLFO M. GONZÁLEZ-LEBRERO; MARÍA CANDELARIA DE LA FUENTE; EMANUEL E. STREHLER; ROLANDO C. ROSSI; JUAN PABLO F. C. ROSSI

JOURNAL OF BIOLOGICAL CHEMISTRY

AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC

Lugar: Bethesda, Maryland; Año: 2011 p. 32018 - 32025

0021-9258

In this work, we set out to identify and characterize the calciumoccluded 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 occlusionof Ca2
in microsomes containing PMCA. This involves asystem for overexpression of the PMCA and the use of a rapidmixing device combined with a filtration chamber, allowing theisolation 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 dependencewith an apparent dissociation constant of 12  2.2 M, whichagrees with the value found through measurements of PMCAactivity in the absence of calmodulin. When enzyme phosphorylationand the retained calcium were studied as a function oftime in the presence of LaIII (inducing accumulation of phosphoenzymein the E1P state), we obtained apparent rate constantsnot significantly different from each other. Quantificationof EP and retained calcium in steady state yield astoichiometry of one mole of occluded calcium per mole ofphosphoenzyme. These results demonstrate for the first timethat one calcium ion becomes occluded in the E1P-phosphorylatedintermediate of the PMCA.