Human Recombinant Phosphodiesterase 4B2B Binds (R)-Rolipram at a Single Site with Two Affinities

Citations Over TimeTop 14% of 1997 papers

Abstract

The interactions between (R)-rolipram and purified human recombinant low-Km, cAMP-specific phosphodiesterase (HSPDE4B2B) constructs were investigated using biochemical, kinetic, and biophysical approaches. The full-length protein (amino acids 1-564) and an N-terminal truncated protein (amino acids 81-564) exhibited high-affinity (R)-rolipram binding, whereas an N-terminal and C-terminal truncated protein (amino acids 152-528) lacked high-affinity (R)-rolipram binding. The 152-528 and 81-564 proteins had similar Km's and kcat/Km's and differed less than 4-fold compared with the 1-564 protein. (R)-Rolipram inhibition plots were biphasic for the 1-564 and 81-564 proteins and fit to two states, a high-affinity (Ki = 5-10 nM) state and a low-affinity (Ki = 200-400 nM) state, whereas the 152-528 protein fit to a single state (Ki = 350-400 nM). The stoichiometry for high-affinity binding using a filter binding assay was found to be <1 mol of (R)-rolipram per mole of 1-564 or 81-564 protein. Titration microcalorimetric studies revealed both a high-affinity state with a stoichiometry of 0.3 mol of (R)-rolipram per mole of protein and a low-affinity state with a stoichiometry of 0.6 mol of (R)-rolipram per mole of protein for the 81-564 protein. A single low-affinity state with a stoichiometry of 0.9 mol of (R)-rolipram per mole of protein was seen using the 152-528 protein. The data indicate that purified HSPDE4B2B 1-564 and 81-564 proteins contain a single binding site for (R)-rolipram and suggest that the proteins exist in two different states distinguishable by their affinity for (R)-rolipram. Furthermore, the high-affinity binding state of the protein requires amino acid residues at the N-terminus (81-151) of the protein and catalytic domain (152-528), whereas the low-affinity binding state only requires residues in the catalytic domain (152-528). Phosphorylation at residues 487 and 489 of the 81-564 protein does not appear to alter the substrate kinetics or the stoichiometry and binding affinity of (R)-rolipram.

Related Papers

• → Acute hypoxia modifies cAMP levels induced by inhibitors of phosphodiesterase‐4 in rat carotid bodies, carotid arteries and superior cervical ganglia(2010)15 cited
• → Inhibitory Effects of Rolipram on Partially Purified Phosphodiesterase 4 From Rat Brains(1998)7 cited
• → Phosphodiesterase type 4 that regulates cAMP level in cortical neurons shows high sensitivity to rolipram(1997)26 cited
• → Tissue specificity of cAMP‐phosphodiesterase inhibitors: Rolipram, amrinone, milrinone, enoximone, piroximone, and imazodan(1988)12 cited
• → Structure and Function of the High Affinity, Rolipram and RO 2-1724 Sensitive, cAMP Phosphodiesterases(1990)