DETAILED CHARACTERIZATION OF A PURIFIED TYPE-4 PHOSPHODIESTERASE, HSPDE4B2B - DIFFERENTIATION OF HIGH-AFFINITY 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 rol ipram-sensitive cAMP phosphodiesterase, HSPDE4B2B (amino acid residues 81-564). The protein expression levels were approximately 8 mg of HSP DE4B2B (81-564) per liter of Sf9 cells. The K-m of the purified enzyme for cAMP was 4 mu M and the K-i for the Type 4 phosphodiesterase-spec ific inhibitor (R)-rolipram was 0.6 mu M. The specific activity of the purified protein was 40 mu mol/min/mg protein. A nonequilibrium filte r binding assay revealed a high-affinity (R)-rolipram binding site on the purified enzyme with a K-d of 1.5 nM and a stoichiometry of 0.05-0 .3 mol of (R)-rolipram per mol of HSPDE4B2B (81-564). Equilibrium dial ysis experiments revealed a single binding constant of 140 nM with a s toichiometry of 0.75 mol of (R)-rolipram per mol of HSPDE4B2B (81-564) . Size exclusion chromatography and analytical ultracentrifugation exp eriments suggest that the protein exists in multiple association state s larger than a monomer. Proteolysis experiments revealed a 43-kDa fra gment that contained catalytic and rolipram-inhibitable activities, bu t the fragment showed no high-affinity (R)-rolipram binding. Based on the proteolytic cleavage studies a 43-kDa protein was constructed, exp ressed, and purified. This protein, HSPDE4B2B (152-528), had K-m and V -max similar to those of the HSPDE4B2B (81-564) protein, but did not e xhibit high-affinity (R)-rolipram binding. The protein did show low-af finity (R)-rolipram binding using the equilibrium binding assay. These results show that a low-affinity binding site for (R)-rolipram is sol ely contained within the catalytic domain of HSPDE4B2B, whereas high-a ffinity (R)-rolipram binding requires residues within the catalytic do main and residues flanking N- and/or C-terminal to the catalytic regio n. (C) 1997 Academic Press.