Purification and characterization of the human PDE4A catalytic domain (PDE4A330-723) expressed in Sf9 cells

The human PDE4A catalytic domain (PDE4A 330–723 ) expressed in Sf9 cells was found to be heavily phosphorylated on both serines of the conserved SPS motif by mass spectrometric analysis. The purified protein exists as a tetramer at a concentration ∼1 mg/ml from light scattering measurement and has a K m of 2 μM in hydrolyzing cAMP. In comparison, a partially purified PDE4A 330–723 expressed in Escherichia coli has an apparent K m of 10 μM. The EC 50 values for the Mg 2+ - or Co 2+ -mediated cAMP hydrolysis between the two enzymes differed by less than twofold. In addition, both enzymes exhibit similar sensitivities toward inhibition by a diverse set of inhibitors. Together with the fact that its adjacent peptide was covalently labeled by an electrophilic cAMP analogue, these results support that the SPS motif is not part of but is positioned near the active site. An efficient purification protocol that provides a highly purified PDE4A catalytic domain suitable for crystallization study is described.