Refolding and kinetic characterization of the phosphodiesterase-8A catalytic domain.
Cyclic nucleotide phosphodiesterase-8 (PDE8) hydrolyzes the second messenger cAMP and is involved in many biological processes such as testosterone production. Although the bacterial and mammalian expression systems have been extensively tried, production of large quantity of soluble and active PDE8 remains to be a major hurdle for pharmacological and structural studies. Reported here is a detailed protocol of refolding and purification of large quantity of the PDE8A1 catalytic domain (residues 480-820) and kinetic characterization of the refolded protein. This protocol yielded about 8 mg of the PDE8A catalytic domain from 2l Escherichia coli culture, which has at least 40-fold higher activity than those reported in literature. The PDE8A1 catalytic domain has k(cat) of 4.0 s(-1) for Mn(2+) and 2.9s(-1) for Mg(2+), and the K(M) values of 1-1.8 microM. In addition, the PDE8A1 (205-820) fragment that contains both PAS and catalytic domains was expressed in E. coli and refolded. This PDE8A1 (205-820) fragment has k(cat) of 1.1 s(-1) and K(M) of 0.28 microM, but aggregated at high concentration. The K(M) of PDE8A1 (205-820) is 2- to 7-fold higher than the K(M) values of 40-150 nM for the full-length PDE8s in literature, but about 6-fold lower than that of the catalytic domain. Thus, the K(M) difference likely implies an allosteric regulation of the PDE8A activity by its PAS domain.
Yan
Zier
Z
Laboratory of Structure Biology, School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510275, PR China.
Wang
Huanchen
H
Cai
Jiwen
J
Ke
Hengming
H
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D020134:Catalytic Domain; D004926:Escherichia coli / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D020673:Protein Renaturation; D017434:Protein Structure, Tertiary; D013696:Temperature
D010446:Peptide Fragments; D015105:3',5'-Cyclic-AMP Phosphodiesterases; C112770:PDE8A protein, human