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

doi:10.1006/prep.1996.0683

Protein Expression and Purification

Volume 9, Issue 2, March 1997, Pages 191-202

https://doi.org/10.1006/prep.1996.0683 Get rights and content

Abstract

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. TheK_mof the purified enzyme for cAMP was 4 μmand theK_ifor 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 aK_dof 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), hadK_mandV_maxsimilar 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.

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^☆: P. GreengardG. A. Robison, Eds.

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

Abstract

Trends Pharmacol. Sci.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Gene

Biochim. Biophys. Acta

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

J. Biol. Chem.

Eur. J. Pharmacol.

J. Biol. Chem.

Advances in Second Messenger and Phosphoprotein Research

Identification of a conserved domain among cyclic nucleotide phosphodiesterases from diverse species

Proc. Natl. Acad. Sci.

Calmodulin-stimulated cyclic nucleotide phosphodiesterases

A family of human phosphodiesterases homologous to thedunceDrosophila melanogaster

Mol. Cell. Biol.

Phosphodiesterase inhibitors: New opportunities for the treatment of asthma

Thorax

Isolation of similar rolipram-inhibitable cyclic-AMP-specific phosphodiesterases from rat brain and heart

Eur. J. Biochem.

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