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 similar to1 mg/ml from light scattering measurement and has a K-m of 2 muM in hydrolyzing cAMP. In comparison, a partially purified 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 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. (C) 2001 Academic Press.