INVESTIGADORES
GONZALEZ LEBRERO Rodolfo Martin
congresos y reuniones científicas
Título:
Calcium Occlusion in the Phosphorylated Intermediate of Plasma Membrane Ca2+-ATPase
Autor/es:
MARIELA, FERREIRA GOMES; RODOLFO M GONZÁLEZ LEBRERO; MC DE LA FUENTE; RC. ROSSI; JPFC ROSSI
Lugar:
Salta
Reunión:
Congreso; XXXIX Annual Meeting of the Argentinean Biophysical Society (SAB) and 3rd Latin American Protein Society Meeting; 2010
Institución organizadora:
Sociedad Argentina de Biofísica
Resumen:
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The Plasma Membrane Calcium ATPase
(PMCA) is a calmodulin-regulated P-type ATPase responsible for the
maintenance of low intracellular concentration of Ca2+ in most
eukaryotic cells. The current kinetic model proposes that the enzyme
exists in two main conformations, E1 and E2. After binding of
intracellular Ca2+ to high-affinity sites, E1 can be phosphorylated
by ATP with formation of the intermediate E1P, which would result in
occlusion of bound Ca2+. After a conformational transition to E2P,
Ca2+ would be released to the extracellular medium from low-affinity
sites, the phosphoenzyme is hydrolyzed and the resulting E2
intermediate state undergoes a new conformational transition to E1.
The aim of this work was to identify
the calcium occlusion intermediate and to assess whether the
occlusion of calcium is concomitant with the phosphorylation of the
enzyme by ATP.
We used PMCA obtained by
Baculovirusmediated expression in Sf9 insect cells and isolated as
microsomal membranes. Ca2+ occlusion was measured using the method
described by Rossi et al. (1999)1 with minor modifications.
We measured the amount of the
phosphorylated intermediate and of calcium occluded in the PMCA as a
function of time, under the same experimental conditions, and
obtained similar apparent rate constants. Quantification of EP and
occluded calcium in steady state yield a stoichiometry of one mole of
occluded calcium per mole of phosphoenzyme. Therefore, we can
conclude that the formation of phosphoenzyme and the calcium
occlusion are simultaneous events.