Purification and physical characterization of cloned human cAMP phosphodiesterases PDE-4D and -4C.
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.
Saldou
N
N
Roche Bioscience, Palo Alto, CA 94304, USA.
Baecker
P A
PA
Li
B
B
Yuan
Z
Z
Obernolte
R
R
Ratzliff
J
J
Osen
E
E
Jarnagin
K
K
Shelton
E R
ER
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D016367:Baculoviridae / Q000235:genetics; D002853:Chromatography, Liquid / Q000379:methods; D003001:Cloning, Molecular; D054703:Cyclic Nucleotide Phosphodiesterases, Type 4; D019281:Dimerization; D005822:Genetic Vectors; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D008970:Molecular Weight; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism
D007527:Isoenzymes; D011993:Recombinant Fusion Proteins; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D054703:Cyclic Nucleotide Phosphodiesterases, Type 4