Separation of multiple forms of cyclic nucleotide phosphodiesterases from rat brain by isoelectrofocusing

Multiple forms of cyclic nucleotide phosphodiesterase(s) (EC 3.1.4-) have been investigated using isoelectric focusing techniques. Six distinct peaks of cyclic AMP phosphodiesterase activity are apparent in a 105 000 × g soluble supernatant fraction of sonicated rat cerebellum. These peaks, designated A-F, have isoelectric points (pI values) of 4.4, 4.8, 5.0, 6.1, 8.3 and 9.0, respectively, varying affinities for cyclic AMP and cyclic GMP, different kinetic behavior, and divergent subcellular localization. Peaks B, C and D contain appreciable cyclic GMP phosphodiesterase activities, while Peaks A, E and F hydrolyze little or no cyclic GMP. Kinetic analysis of five of the focused peaks showed non-linear Lineweaver-Burk plots closely approximating those of the original cerebellar homogenate. Discontinuous sucrose gradient fractionation before isoelectric focusing indicates that Peaks B, D and E are cytosolic forms and the others appear particulate in nature. In contrast to multiple forms separated by preparative polyacrylamide gel electrophoresis, activity peaks separated by isoelectric focusing do not respond to an endogenous activator of the cyclic AMP phosphodiesterase activity. Since the isoelectric focusing technique has preparative and analytical advantages over other electrophoretic methods, it provides a good procedure to investigate components of the complex cyclic nucleotide phosphodiesterase enzyme system.