Detailed Characterization of a Purified Type 4 Phosphodiesterase, HSPDE4B2B: Differentiation of High- and Low-Affinity (R)-Rolipram Binding

We have overexpressed in a baculovirus expression system, and purified to >95% homogeneity, milligram quantities of a human recombinant rolipram-sensitive cAMP phosphodiesterase, HSPDE4B2B (amino acid residues 81–564). The protein expression levels were approximately 8 mg of HSPDE4B2B (81–564) per liter of Sf9 cells. TheKmof the purified enzyme for cAMP was 4 μmand theKifor the Type 4 phosphodiesterase-specific inhibitor (R)-rolipram was 0.6 μm. The specific activity of the purified protein was 40 μmol/min/mg protein. A nonequilibrium filter binding assay revealed a high-affinity (R)-rolipram binding site on the purified enzyme with aKdof 1.5 nmand a stoichiometry of 0.05–0.3 mol of (R)-rolipram per mol of HSPDE4B2B (81–564). Equilibrium dialysis experiments revealed a single binding constant of 140 nmwith a stoichiometry of 0.75 mol of (R)-rolipram per mol of HSPDE4B2B (81–564). Size exclusion chromatography and analytical ultracentrifugation experiments suggest that the protein exists in multiple association states larger than a monomer. Proteolysis experiments revealed a 43-kDa fragment that contained catalytic and rolipram-inhibitable activities, but the fragment showed no high-affinity (R)-rolipram binding. Based on the proteolytic cleavage studies a 43-kDa protein was constructed, expressed, and purified. This protein, HSPDE4B2B (152–528), hadKmandVmaxsimilar to those of the HSPDE4B2B (81–564) protein, but did not exhibit high-affinity (R)-rolipram binding. The protein did show low-affinity (R)-rolipram binding using the equilibrium binding assay. These results show that a low-affinity binding site for (R)-rolipram is solely contained within the catalytic domain of HSPDE4B2B, whereas high-affinity (R)-rolipram binding requires residues within the catalytic domain and residues flanking N- and/or C-terminal to the catalytic region.