Isolation and Purification of the Ca2+ Pump

The isolation and purification of the plasma membrane Ca²⁺ pump is essential for detailed structural studies and for the unambiguous identification of those Ca²⁺-dependent enzymatic activities of membrane preparations which are catalyzed by the Ca²⁺ pump. Affinity chromatography is in many cases the procedure of choice for separating components which, like the Ca²⁺-ATPase, are present in minute amounts in complex mixtures. In the sarcoplasmic reticulum of skeletal muscle, up to 70% of the membrane protein is the Ca²⁺-pumping ATPase. The purified Ca²⁺-ATPase can be stored for several weeks at liquid nitrogen temperature in media containing Ca²⁺ and calmodulin without loss in activity. Calmodulin affinity chromatography can be used for the purification of a calmodulin-dependent enzyme if the following two criteria are satisfied. The enzyme has to bind with high affinity to calmodulin. The enzyme has to be the only calmodulin-binding protein in the mixture, since calmodulin-Sepharose columns will retain all substances that bind calmodulin.