Active Transport of Divalent Cations by the Plasma Membrane Calcium Pump.

FERREIRA GOMES MARIELA; MARTIARENA JORGE; ROSSI, J.P.F.C.

ROSARIO, SANTA FE

Congreso; XXXVI Reunión Annual de la Sociedad Argentina de Biofísica; 2006

SOCIEDAD ARGENTINA DE BIOFISICA

  The plasma membrane calcium pump (PMCA) is a fundamental mechanism to actively remove Ca2+ from the cytoplasm of all eukaryotic cells. PMCA, like all members of the superfamily of P-type ATPases catalyzes the hydrolysis of ATP. The main cation transported by PMCA is Ca2+ although reports about other divalent cations (C2+) transport are rather controversial. We report here a method to measure the active transport of C2+ in inside-out vesicles prepared from human red cell membranes. Inside-out vesicles expose ATP and transport sites to the reaction medium and it is therefore possible to measure volume changes due to the transport of divalent cations that drive water into the vesicles. Volume changes were measured recording the intensity of light scattered by a suspension of these vesicles which express mostly the isoform PMCA 4b. Transport of each C2+ was assessed by measuring its concentration in vesicles after 30-50 min pumping by Atomic Absortion. The methodology here described showed that it is possible: (a) to follow the active transport of Ca2+ measuring the slope of light scattering intensity as a function of time; (b) to measure the influence of inhibitors and activators of PMCA; (c) to measure the transport of divalent cations. With grants of ANPCYT, UBACYT and CONICET.