The control by Ca²⁺ of the polyphosphoinositide phosphodiesterase and the Ca²⁺-pump ATPase in human erythrocytes

Both the Ca²⁺pump ATPase and the phosphoinositide phosplhodiesterase of the erythrocyte membrane can, when assayed under appropriate conditions, be activated by Ca²⁺ in the micromolar range. We have therefore compared the mechanisms and affinities for Ca²⁺ activation of the two enzymes in human erythrocyte membranes, to see whether the polyphosphoinositide phosphodiesterase would be active in normal healthy erythrocytes. At physiological ionic strength and in the presence of calmodulin, the Ca²⁺-pump ATPase was activated by Ca²⁺ in a highly co-operative manner, with half-maximal activation occurring at about 0.3 μM-Ca²⁺. At an optimal Ca²⁺ concentration, calmodulin stimulated the Ca²⁺-sensitive ATPase activity about 10-fold. Ca²⁺ activated the polyphosphoinositide phosphodiesterase in a non-cooperative manner. The Ca²⁺ requirements for breakdown of phosphatidylinositol phosphate and phosphatidylinositol 4,5-bisphosphate were identical, which supports our previous conclusion that Ca²⁺ activates a single polyphosphoinositide phosphodiesterase that degrades both lipids with equal facility. Added calmodulin did not affect the activity of the polyphosphoinositide phosphodiesterase. At low ionic strength in the absence of Mg²⁺, half-maximal activation of the phosphodiesterase was at about 3 μM-Ca²⁺. The presence of 1 mM-Mg²⁺ shifted the Ca²⁺ activation curve to the right, as did elevation of the ionic strength. When the Ca²⁺ pump ATPase and the polyphosphoinositide phosphodiesterase were assayed in the same incubations and under conditions of intracellular ionic strength and Mg²⁺ concentration, the ATPase was fully activated at 3 μM-Ca²⁺, whereas no polyphosphoinositide phosphodiesterase activity was detected below 100 μM-Ca²⁺. The Ca²⁺-pump ATPase of the erythrocyte membrane normally maintains the Ca²⁺ concentration of healthy erythrocytes below approx. 0.1 μM. It therefore seems unlikely that the polyphosphoinositide phosphodiesterase of the erythrocyte membrane ever expresses its activity in a healthy erythrocyte.