Apparent activation of Ca2+-ATPase activity by dilution.

VANAGAS L.; ROSSI R.C.; ROSSI J.P.F.C.

Bariloche

Congreso; XXXIX Reunión anual de la SAiB; 2003

Ca2+ pumps (PMCAs) are widely distributed among different tissues. Generically the pump is a single polypeptide chain of 127,000 to 137,000 daltons. PMCA is a calmodulin-regulated P-type ATPase that is encoded by a multigene family. To characterize the mechanism of PMCA under different conditions, it is necessary to measure Ca2+-ATPase activity, Ca2+ transport and the partial reactions like, phospho and dephosphorylation and occlusion of Ca2+. We have found that specific activity of Ca2+-ATPase of erythrocyte membranes, measured at concentrations below 50 mg/ml of protein, increases steeply up to 3-5 times at concentrations of 1 mg/ml of total membrane protein. Dilution of the protein did not modify the activation by ATP, Ca2+ or Ca2+-calmodulin. This apparent activation of the enzyme is not affected by ionic strength and is independent of the method of Pi measurement (colorimetric or determination of release of g32P-ATP). The apparent activation on the enzyme is mimicked by 0.0025-0.01% of detergent C12E10 and is enhanced when membranes were slowly frozen instead of frozen by immersion in liquid nitrogen. This behavior is also observed in microsomal preparations of h4b PMCA expressed in insect Sf9 cells. These results seem to be accounted for by two reasons: a) dilution reveals sites hidden by high lipid-protein concentrations or b) dilution reveals the action of a modulator like FXYD proteins in Na,K-ATPase.