title: Calcium occlusion in plasma membrane Ca2+-ATPase.
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(1)P 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(1)P-phosphorylated intermediate of the PMCA.
authors.affiliation: Instituto de Química y Fisicoquímica Biologicas, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Consejo Nacional de Investigaciones Científicas y Técnicas, Junín 956, 1113 Buenos Aires, Argentina.
authors.forename: Mariela S
authors.initials: MS
authors.lastname: Ferreira-Gomes
authors.forename: Rodolfo M
authors.initials: RM
authors.lastname: González-Lebrero
authors.forename: María C
authors.initials: MC
authors.lastname: de la Fuente
authors.forename: Emanuel E
authors.initials: EE
authors.lastname: Strehler
authors.forename: Rolando C
authors.initials: RC
authors.lastname: Rossi
authors.forename: Juan Pablo F C
authors.initials: JP
authors.lastname: Rossi
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D006801:Humans; D017136:Ion Transport / Q000502:physiology; D010766:Phosphorylation / Q000502:physiology; D053499:Plasma Membrane Calcium-Transporting ATPases / Q000235:genetics / Q000378:metabolism*; D018411:Spodoptera
chemical_list: C507517:ATP2B4 protein, human; D053499:Plasma Membrane Calcium-Transporting ATPases; D002118:Calcium