[20] Use of Calmodulin Affinity Chromatography for Purification of Specific Calmodulin-Dependent Enzymes

This chapter discusses the preparation of the calmodulin (CaM)-affinity column and its general application for the purification of CaM-regulated proteins. Owing to the large number of CaM-regulated proteins contained in most tissue sources, CaM-Sepharose 4B affinity column chromatography is not sufficient by itself for the purification of a specific enzyme to homogeneity. However, in combination with additional protein purification techniques, several homogeneous CaM-dependent enzymes and proteins are obtained. The purification of bovine brain CaM-dependent cyclic nucleotide phosphodiesterase and calcineurin is presented as an example in the chapter. Two procedures have been used in the preparation of CaM-Sepharose 4B: one uses the cyanogen bromide-activated gel and the other uses a divinyl sulfone activation method. Because CaM is multifunctional, many enzymes and proteins are capable of interacting with the CaM-Sepharose 4B column. Therefore, CaM-affinity chromatography is not sufficient by itself to purify one enzyme to homogeneity. However, several CaM-regulated proteins have been purified using CaM-Sepharose 4B affinity chromatography in combination with other purification steps. These include CaM-dependent phosphodiesterase, calcineurin, myosin light-chain kinase, phosphorylase kinase, erythrocyte Ca²⁺, Mg²⁺-ATPase, calmodulin-binding protein II, plant NAD kinase, and adenylate cyclase.