Table 4

Ligand binding characteristics of H505N, H506N, and hrPDE4A/Met201-886

Met201-886H505NH506N
KD (nm)
 (R)-Rolipram1.3  ± 0.51.8  ± 0.43.9  ± 0.9
 RP 734010.2  ± 0.040.3  ± 0.10.5  ± 0.1
B max (pmol/ml)
 (R)-Rolipram1.0  ± 0.10.8  ± 0.20.3  ± 0.05
 RP 734012.4  ± 0.32.1  ± 0.11.0  ± 0.1
IC50 cAMP (μm)
 [3H]-(R)-Rolipram49  ± 868  ± 23626  ± 196
 [3H]RP 7340142  ± 1278  ± 12552  ± 129

Saturation binding experiments were conducted using ligand concentrations of 0.2–52 nm[3H]-(R)-rolipram or 0.2–10 nm[3H]RP 73401 in the filtration assay as described in the text. Scatchard analyses were performed using the Acufit computer program. Ligand concentrations for competition binding studies were 1 nm [3H]RP 73401 and 2 nm[3H]-(R)-Rolipram. IC50 values were calculated using the Cheng-Prusoff equation (40): IC50= IC50/[ligand]/(Kd + 1). Data were normalized for different expression levels of PDE4A protein by immunoblot analysis using rhPDE4A/Met201-886 as a standard. Data represent the mean ± standard error of three to six separate experiments.