Calcium occlusion in plasma membrane Ca 2+-ATPase

Mariela S. Ferreira-Gomes, Rodolfo M. González-Lebrero, María C. De La Fuente, Emanuel E. Strehler, Rolando C. Rossi, Juan Pablo F.C. Rossi

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10 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)32018-32025
Number of pages8
JournalJournal of Biological Chemistry
Volume286
Issue number37
DOIs
StatePublished - Sep 16 2011

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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    Ferreira-Gomes, M. S., González-Lebrero, R. M., De La Fuente, M. C., Strehler, E. E., Rossi, R. C., & Rossi, J. P. F. C. (2011). Calcium occlusion in plasma membrane Ca 2+-ATPase. Journal of Biological Chemistry, 286(37), 32018-32025. https://doi.org/10.1074/jbc.M111.266650