Measuring cAMP Specific Phosphodiesterase Activity: A Two-step Radioassay.

Cyclic nucleotide degrading phosphodiesterase (PDE) enzymes are crucial to the fine tuning of cAMP signaling responses, playing a pivotal role in regulating the temporal and spatial characteristics of discrete cAMP nanodomains and hence the activity of cAMP-effector proteins. As a consequence of orchestrating cAMP homeostasis, dysfunctional PDE activity plays a central role in disease pathogenesis. This highlights the need for developing methods that can be used to further understand PDE function and assess the effectiveness of potentially novel PDE therapeutics. Here we describe such an approach, where PDE activity is indirectly measured through the direct quantification of radioactively tagged cAMP (pmol/min-1/mg-1). This method provides a highly sensitive tool for investigating PDE functionality.