Assay and Purification of Calmodulin-Dependent Cyclic Nucleotide Phosphodiesterase and Isozyme Separation

In general, the action of intracellular second messengers, 3′:5′-cyclic adenosine monophosphate (cAMP) and 3′:5′-cyclic guanosine monophosphate (cGMP), are terminated by cyclic nucleotide phosphodiesterase (PDE). In most mammalian tissues, PDE exists in multiple forms that differ in subcellular localization, catalytic, regulatory, and immunological properties (1 –4 ). One of these forms of enzyme is activated by Ca²⁺ and calmodulin (CaM) and has a lower apparent K _m for cGMP than for cAMP and higher V _max for cAMP than for cGMP (2 ,5 ,6 ). This CaM-dependent cyclic nucleotide phosphodiesterase (CaMPDE) is widely distributed among mammalian tissues and other eukaryotes (7 –11 ). CaMPDE is one of the most intensively studied and best characterized PDEs (12 –19 ).