Purification and characterization, of the human PDE4A catalytic domain (PDE4A(330-723)) expressed in Sf9 cells

The human PDE4A catalytic domain (PDE4A(330-723)) expressed in Sf9 cells wa s found to be heavily phosphorylated on both serines of the conserved SPS m otif by mass spectrometric analysis. The purified protein exists as a tetra mer at a concentration similar to1 mg/ml from light scattering measurement and has a K-m of 2 muM in hydrolyzing cAMP. In comparison, a partially puri fied PDE4A(330-723) expressed in Escherichia coli has an apparent K-m of 10 muM The EC50 values for the Mg2+- or Co2+-mediated cAMP hydrolysis between the two enzymes differed by less than twofold. In addition, both enzymes e xhibit similar sensitivities toward inhibition by a diverse set of inhibito rs. Together with the fact that its adjacent peptide was covalently labeled by an electrophilic cAMP analogue, these results support that the SPS moti f is not part of but is positioned near the active site. An efficient purif ication protocol that provides a highly purified PDE4A catalytic domain sui table for crystallization study is described. (C) 2001 Academic Press.