22] Isolation and characterization of bovine brain calcineurin: A calmodulin-stimulated protein phosphatase

Abstract

This chapter focuses on the isolation and characterization of the bovine brain calcineurin. Calcineurin is a heterodimer composed of two subunits, a 19,000 Mr Ca2+-binding subunit, calcineurin B, and a 61,000 Mr subunit, calcineurin A, which interacts with calmodulin. A Ca2+-dependent, calmodulin-stimulated protein phosphatase activity has been found to be associated with calcineurin. The protein can be purified on the basis of its ability to interact with calmodulin after sodium dodecyl sulphate- (SDS)-gel electrophoresis, as well as by its protein phosphatase activity. Upon SDS-polyacrylamide gel electrophoresis, the small subunit of calcineurin, calcineurin B, can be readily identified as a small polypeptide with an apparent Mr between 16,000 and 15,000 that exhibits a characteristic increase in mobility in the presence of Ca2+. Once identified, this subunit can be quantitated by densitometric analysis of slab gels stained with Coomassie Brilliant Blue. The 61,000 Mr subunit, calcineurin A, is more difficult to identify on Coomassie Blue-stained gels of crude protein mixtures because of the presence of many polypeptides of similar molecular weight. However, calcineurin A, the calmodulin-binding subunit of calcineurin, can interact with calmodulin even after SDS-gel electrophoresis and it can be identified and quantitated by a modification of the [125I]calmodulin gel overlay technique.