Purification and characterization of the human PDE4A catalytic domain (PDE4A330-723) expressed in Sf9 cells.
The human PDE4A catalytic domain (PDE4A330-723) 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 approximately 1 mg/ml from light scattering measurement and has a Km of 2 microM in hydrolyzing cAMP. In comparison, a partially purified PDE4A330-723 expressed in Escherichia coli has an apparent Km of 10 microM. 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.
Lario
P I
PI
Department of Biochemistry, McGill University, 3655 Promenade Sir-William-Osler, Room 802, McIntyre Medical Sciences Building, Montreal, Quebec, H3G 1Y6, Canada.
Bobechko
B
B
Bateman
K
K
Kelly
J
J
Vrielink
A
A
Huang
Z
Z
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D020816:Amino Acid Motifs; D000818:Animals; D020134:Catalytic Domain; D002413:Cations, Divalent / Q000378:metabolism; D002460:Cell Line; D002850:Chromatography, Gel; D003067:Coenzymes / Q000378:metabolism; D000242:Cyclic AMP / Q000378:metabolism; D054703:Cyclic Nucleotide Phosphodiesterases, Type 4; D004791:Enzyme Inhibitors / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D006868:Hydrolysis; D010766:Phosphorylation; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization; D018411:Spodoptera
D002413:Cations, Divalent; D003067:Coenzymes; D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D000242:Cyclic AMP; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D054703:Cyclic Nucleotide Phosphodiesterases, Type 4; C517355:PDE4A protein, human