28] Chemical approaches to the calmodulin system

Abstract

The ability of calmodulin to make Ca2+-dependent complexes with target proteins and some small molecule ligands, a property dictated by its structural features, has provided essential tool for various types of studies. Many of these employ chemically modified derivatives of calmodulin that retain some or all activity. Detailed information concerning the highly conserved nature of the calmodulin molecule and its characterization, as well as the preparation of specific, functional calmodulin derivatives, is important to all researchers interested in the studies of Ca2+-dependent regulatory phenomena. This chapter reviews the way the techniques of protein chemistry can be applied to the studies of the calmodulin system. The characterization of calmodulin stocks should consist of some basic steps: (1) the examination of UV absorption spectrum; (2) polyacrylamide gel electrophoretic (PAGE) analysis in the presence and absence of denaturants; (3) the assay for Ca2+-dependent functional properties; and (4) amino acid analysis of acid hydrolyzed aliquots. The latter allows not only a test for gross contamination, but also provides the best method for accurate determination of the concentration of calmodulin concentrations.