Coexpression of human cAMP-specific phosphodiesterase activity and high affinity rolipram binding in yeast

• Published in: The Journal of biological chemistry 1992-01, Vol.267 (3), p.1798-1804
• Main Authors: Torphy, T J, Stadel, J M, Burman, M, Cieslinski, L B, McLaughlin, M M, White, J R, Livi, G P
• Format: Article
• Language: English
• Subjects: 3',5'-Cyclic-AMP Phosphodiesterases - genetics; 3',5'-Cyclic-AMP Phosphodiesterases - isolation & purification; 3',5'-Cyclic-AMP Phosphodiesterases - metabolism; Analytical, structural and metabolic biochemistry; Binding, Competitive; Biological and medical sciences; Chromatography, Ion Exchange; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; Gene Expression; Humans; Hydrolases; Immunoblotting; Kinetics; Molecular Weight; Monocytes - enzymology; Phosphodiesterase Inhibitors - metabolism; Plasmids; Purinones - pharmacology; Pyrrolidinones - metabolism; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; Rolipram; Saccharomyces cerevisiae; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(18)46016-4

Studies by various investigators have demonstrated that the low Km, cAMP-specific phosphodiesterase (PDE IV) is selectively inhibited by a group of compounds typified by rolipram and Ro 20-1724. In addition to inhibiting the catalytic activity of PDE IV, rolipram binds to a high affinity binding site present in brain homogenates. Although it has been assumed that the high affinity rolipram-binding site is PDE IV, no direct evidence has been produced to support this assumption. The present studies were undertaken to determine whether the rolipram-binding site is coexpressed with PDE IV catalytic activity in Saccharomyces cerevisiae genetically engineered to express human recombinant monocytic PDE IV (hPDE IV). Expressing hPDE IV cDNA in yeast resulted in a 20-fold increase in PDE activity that was evident within 1 h of induction and reached a maximum by 3-6 h. The recombinant protein represented hPDE IV as judged by its immunoreactivity, molecular mass (approximately 88 kDa), kinetic characteristics (cAMP Km = 3.1 microM; cGMP Km greater than 100 microM), sensitivity to rolipram (Ki = 0.06 microM), and insensitivity to siguazodan (PDE III inhibitor) and zaprinast (PDE V inhibitor). Saturable, high affinity [3H] (R)-rolipram-binding sites (Kd = 1.0 nM) were coexpressed with PDE activity, indicating that both binding activity and catalytic activity are properties of the same protein. A limited number of compounds were tested for their ability to inhibit hPDE IV catalytic activity and compete for [3H](R)-rolipram binding. Analysis of the data revealed little correlation (r2 = 0.35) in the structure-activity relationships for hPDE IV inhibition versus competition for [3H] (R)-rolipram binding. In fact, certain compounds (e.g. (R)-rolipram Ro 20-1724) possessed a 10-100-fold selectivity for inhibition of [3H] (R)-rolipram binding over hPDE IV inhibition, whereas others (e.g. dipyridamole, trequinsin) possessed a 10-fold selectivity for PDE inhibition. Thus, although the results of these studies demonstrate that hPDE IV activity and high affinity [3H](R)-rolipram binding are properties of the same protein, they do not provide clear cut evidence linking the binding site with the PDE inhibitory activity of rolipram and related compounds.