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Purification and Characterization of Phosphodiesterase 4B
Author(s) -
Tam May S.K.,
Liu Yan,
Malakian Karl,
Aulabaugh Ann,
Johnson Mark,
Svenson Kristine,
Ghavami Afshin,
Parris Kevin,
Failli Amedeo,
Kriz Ron,
Stahl Mark,
Lin Laura,
Wen Yingxia
Publication year - 2007
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.21.6.lb26
Subject(s) - creb , phosphodiesterase , phosphorylation , chemistry , protein kinase a , cyclic nucleotide phosphodiesterase , microbiology and biotechnology , creb1 , kinase , second messenger system , biochemistry , transcription factor , signal transduction , biology , enzyme , gene
Cyclic nucleotide phosphodiesterases (PDEs) play critical roles in maintaining the cellular concentration cGMP and cAMP. These key secondary messengers modify the activation of cyclic nucleotide dependent protein kinases that phosphorylate various substrates including ion channels and transcription factors to regulate a myriad of physiological processes. PDE4 is a cAMP specific phosphodiesterase which has been suggested to play a key role in the regulation of PKA/CREB pathway. The PKA/CREB pathway impacts a wide range of physiological responses, including those involved in learning and memory. Inhibition of PDE4 is therefore an attractive approach to development of novel therapies for neurodegenerative disease such as Alzheimer’s disease. To discover novel potent inhibitors of PDE4, we have expressed and purified the catalytic domain of human PDE4B. The purified protein was analyzed by N‐terminal sequencing, mass spectral analysis and stability studies. Our findings indicated that PDE4B catalytic domain was unstable and partially degraded to a smaller more stable conformation. The purified PDE4B preparation, therefore, consisted of a mixture of intact and degraded protein, which may be problematic for activity and crystallization studies. We developed a scheme to generate a stable PDE4B catalytic domain by driving the degradation to completion. The resulting preparation was homogenous and of high specific activity. This high quality PDE4B protein was used for binding assays and structural studies.