TY - JOUR
T1 - Calcium occlusion in plasma membrane Ca 2+-ATPase
AU - Ferreira-Gomes, Mariela S.
AU - González-Lebrero, Rodolfo M.
AU - De La Fuente, María C.
AU - Strehler, Emanuel E.
AU - Rossi, Rolando C.
AU - Rossi, Juan Pablo F.C.
PY - 2011/9/16
Y1 - 2011/9/16
N2 - In this work, we set out to identify and characterize the calcium occluded intermediate(s) of the plasma membrane Ca 2+-ATPase (PMCA) to study the mechanism of calcium transport. To this end, we developed a procedure for measuring the occlusion of Ca 2+ in microsomes containing PMCA. This involves a system for overexpression of the PMCA and the use of a rapid mixing 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 Ca 2+concentration in steady state showed a hyperbolic dependence with an apparent dissociation constant of 12 ± 2.2 μM, 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 La III (inducing accumulation of phosphoenzyme in the E 1P 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 E 1P- phosphorylated intermediate of the PMCA.
AB - In this work, we set out to identify and characterize the calcium occluded intermediate(s) of the plasma membrane Ca 2+-ATPase (PMCA) to study the mechanism of calcium transport. To this end, we developed a procedure for measuring the occlusion of Ca 2+ in microsomes containing PMCA. This involves a system for overexpression of the PMCA and the use of a rapid mixing 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 Ca 2+concentration in steady state showed a hyperbolic dependence with an apparent dissociation constant of 12 ± 2.2 μM, 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 La III (inducing accumulation of phosphoenzyme in the E 1P 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 E 1P- phosphorylated intermediate of the PMCA.
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U2 - 10.1074/jbc.M111.266650
DO - 10.1074/jbc.M111.266650
M3 - Article
C2 - 21795697
AN - SCOPUS:80052725375
SN - 0021-9258
VL - 286
SP - 32018
EP - 32025
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 37
ER -