54] Isolation of bovine brain calmodulin-dependent cyclic nucleotide phosphodiesterase isozymes

Abstract

This chapter describes the procedures for the demonstration and isolation of bovine brain calmodulin (CaM)-dependent phosphodiesterase isozymes. Purified bovine brain CaM-dependent cyclic nucleotide phosphodiesterase exhibits essentially a single protein band on neutral SDS-PAGE gel but shows two protein bands of apparent M r 60,000 and 63,000 on alkaline SDS-PAGE gel. Using this enzyme preparation as the antigen, seven antiphosphodiesterase monoclonal antibodies have been produced and purified. Western immunoblotting analysis has shown that these antibodies may be divided into two groups, one specific toward the 60-kDa polypeptide and the other reacting with both 60-kDa and the 63-kDa polypeptides. A procedure is developed to use these antibodies to demonstrate that the polypeptides represent subunits of different phosphodiesterase isozymes. The chapter summarizes some of the kinetic and regulatory properties of the isolated CaM-dependent cyclic nucleotide phosphodiesterase isozymes from bovine brain. Both isozymes can rise either cAMP or cGMP as substrates and both exhibit higher affinity for cGMP than for cAMP.