[5] Purification and characterization of Acanthamoeba calcium-binding proteins

This chapter focuses on the isolation and characterization of calmodulin from the protozoan Acanthamoeba castellanii and presents a case study of several techniques employed in the initial analysis of calmodulin from a new species. The major consideration during purification must be the limitation of proteolysis. The most difficult step in purifying Acanthamoeba calmodulin is the separation of the whole protein from several 13- to 15-kDa proteolytic fragments. Thus, yield has been sacrificed for speed of isolation in many steps. All operations, except reversed-phase FPLC, are performed on ice or at 4o. The resolution of Acanthamoeba calmodulin peptides by reversed-phase (RP)-HPLC followed by amino acid composition and partial sequence analysis showed a lower level of homology between Acanthamoeba calmodulin and mammalian calmodulin as well as uniqueness of the 14-kDa CaBP. Immunological studies are also employed to assay the degree of similarity among Acanthamoeba calmodulin, other calmodulins, and rabbit skeletal-muscle troponin C. Photocross-linking studies using 8'-azido-ATP with the aim of elucidating the function of the calmodulin-binding proteins are illustrated.