Decrease of apparent calmodulin affinity of erythrocyte ( [formula omitted] at low Ca 2+ concentrations

Abstract

The calmodulin activation of the ( Ca 2+ + Mg 2+)- ATPase (ATP phosphohydrolase, EC 3.6.1.3) in human erythrocyte membranes was studied in the range of 1 nM to 40 μM of purified calmodulin. The apparent calmodulin-affinity of the ATPase was strongly dependent on Ca 2+ and decreased approx. 1000-times when the Ca 2+ concentration was reduced from 112 to 0.5 μM. The data of calmodulin (Z) activation were analyzed by the aid of a kinetic enzyme model which suggests that 1 molecule of calmodulin binds per ATPase unit and that the affinities of the calcium-calmodulin complexes (Ca iZ) decreases in the order of Ca 3 Z > Ca 4 Z > Ca 2 Z ⩾ CaZ . Furthermore, calmodulin dissociates from the calmodulin-saturated Ca 2+-ATPase in the range of 10 −7–10 −6 M Ca 2+, even at a calmodulin concentration of 5 μM. The apparent concentration of calmodulin in the erythrocyte cytosol was determined to be 3 to 5 μM, corresponding to 50–80-times the cellular concentration of Ca 2+-ATPase, estimated to be approx. 10 nmol/g membrane protein. We therefore conclude that most of the calmodulin id dissociated from the Ca 2+-transport ATPase in erythrocytes at the prevailing Ca 2+ concentration (probably 10 −7 – 10 −8 M) in vivo, and that the calmodulin-binding and subsequent activation of the Ca 2+-ATPase requires that the Ca 2+ concentration rises to 10 −6 – 10 −5 M.