Ca 2+-ATPase from chicken ( Gallus domesticus) erythrocyte plasma membrane: effects of calmodulin and taurine on the Ca 2+-dependent ATPase activity and Ca 2+ uptake

Abstract

In the present report we describe a method for purifying plasma membranes from chicken erythrocytes using sonication under conditions that facilitate preferential lysis of plasma membrane, followed by centrifugation through a sucrose gradient. The Ca 2+-dependent ATP hydrolysis by plasma membranes is activated by nanomolar levels of calmodulin, similarly to that from anucleated erythrocytes. Inside-out vesicles display a calmodulin-activated Ca 2+ uptake. Purified Ca 2+-ATPase is obtained from the plasma membrane by Sepharose-calmodulin affinity chromatography, and exhibits an apparent molecular mass of 150 kDa on SDS-polyacrylamide gel electrophoresis, clearly showing that the enzyme is distinct from that described in anucleated erythrocytes (140 kDa). The enzyme is insensitive to physiological concentrations of taurine, a β-amino acid that has been proposed to be involved in Ca 2+ homeostasis of nucleated erythrocytes, suggesting that the effect of taurine is not mediated by the Ca 2+-ATPase. Taken together, these data suggest that the enzyme may be an isoform that resembles the previously described plasma membrane Ca 2+-ATPase from anucleated erythrocytes in its regulation by calmodulin, but differs in its apparent molecular weight.