Figure 4
8-[ϕ-575]-cAMP is a potent PKA activator in vitro. For determination of apparent activation constants, purified recombinant R subunits were allowed to form holoenzyme complexes with PKA-Cα (20 nM) as detailed in the methods section. Activation assays were performed by increasing (0.3 nM-10 μM) cAMP. To obtain apparent activation constants (Kact), the normalized activity of PKA-Cα was plotted against the logarithm of the cAMP (■) and 8-[ϕ-575]-cAMP (▲) concentration and fitted according to a sigmoid dose-response model (Graphpad Prism, variable slope). Each data point represents the mean ± S.D. of two measurements. Experiments were repeated two to three times with similar results.