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. The Km of the purified enzyme for cAMP was 4 microM and the Ki for the Type 4 phosphodiesterase-specific inhibitor (R)-rolipram was 0.6 microM. The specific activity of the purified protein was 40 mumol/min/mg protein. A nonequilibrium filter binding assay revealed a high-affinity (R)-rolipram binding site on the purified enzyme with a Kd of 1.5 nM and 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 nM with 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), had Km and Vmax similar 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.
Rocque
W J
WJ
Department of Molecular Sciences, Glaxo Wellcome, Inc., Research Triangle Park, North Carolina 27709, USA.
Holmes
W D
WD
Patel
I R
IR
Dougherty
R W
RW
Ittoop
O
O
Overton
L
L
Hoffman
C R
CR
Wisely
G B
GB
Willard
D H
DH
Luther
M A
MA
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D016367:Baculoviridae / Q000235:genetics; D001665:Binding Sites; D003001:Cloning, Molecular; D005822:Genetic Vectors / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D006801:Humans; D007700:Kinetics; D008970:Molecular Weight; D010446:Peptide Fragments / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011760:Pyrrolidinones / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins; D020889:Rolipram
D010446:Peptide Fragments; D011760:Pyrrolidinones; D011994:Recombinant Proteins; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D020889:Rolipram