PDE5 Activity Assay

PA protein was assayed for cGMP-hydrolytic activity using a commercially available kit (Enzo, Farmingdale, NY) as previously described (25). Briefly, PA protein was purified over a Centri-Spin 10 column to remove any free phosphates (Princeton Separations, Adelphia, NJ). Purified protein concentrations were determined using the Bradford method (44). Samples were assayed in duplicate for cGMP-hydrolytic activity using a commercially available colorimetric cyclic nucleotide phosphodiesterase assay kit (Enzo, Farmingdale, NY) with and without sildenafil (100nM, Sigma-Aldrich, St. Louis, MO). Results are shown as PDE5-specific pmol cGMP hydrolyzed/min/mg total protein.