Purification and physical characterization of cloned human cAMP phosphodiesterases PDE-4D and-4C |
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Authors: | Natalie Saldou Preston A. Baecker Bin Li Zhengyu Yuan Rena Obernolte James Ratzliff Eric Osen Kurt Jarnagin Earl R. Shelton |
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Affiliation: | (1) Roche Bioscience, 94304 Palo Alto, CA;(2) Biotech, Sunnyvale, CA;(3) Genentech, South San Francisco, CA;(4) Kowa Research Institute, San Jose, CA;(5) Versicor, South San Francisco, CA |
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Abstract: | Individual isozymes of family four cyclic-nucleotide phosphodiesterases (PDE-4s) were characterized and compared in order to advance our understanding of how PDE-4s regulate cAMP levels in cells. Full-length and shorter clones containing various functional domains were constructed and overexpressed using a recombinant baculovirus-infected Sf9 insect cell system. One form each of PDE-4C and 4D was purified 125- and 534-fold, respectively, using anion-exchange and affi-gel blue chromatography. The purified material was unaltered in size on SDS-polyacrylamide gels during purification and nearly homogeneous (>95%) as estimated by both staining and immunoblotting. Approximately 1 mg of PDE-4D (74.7 kDa) and 3.7 mg of PDE-4C (61.4 kDa) could be isolated from a 6-L culture of cells. The physical characteristics of Stokes' radius and sedimentation coefficient for PDE-4 enzymes cloned from each of the four isogenes were determined using size-exclusion chromatography and sedimentation in glycerol gradients. Calculations indicate that both long and short forms can form dimers, although evidence for monomers and higher-order subunit association was seen. Furthermore, the results clearly show that all long and short forms of PDE-4 are highly asymmetric molecules. This work has shown that large amounts of PDE-4 proteins can be purified and characterized physically and enzymatically to yield information that will enable a greater understanding of how PDE-4 enzymes function in cells. |
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Keywords: | Baculovirus cyclic nucleotide phosphodiesterase heterologous expression recombinant protein rolipram |
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