Recombinant expression of a type IV, cAMP-specific phosphodiesterase: characterization and structure-function studies of deletion mutants.
A potential role for cAMP in regulating the differentiation of myoblasts has led us to examine the components of the cAMP signaling system, including the type IV, cAMP-specific phosphodiesterases. The full coding sequence of the phosphodiesterase PDE4D1 was inserted in the bacterial expression vector pGEX-KG. N- and C-terminal truncations were also placed in the same vector, allowing the expression and purification of glutathione S-transferase (GST)-PDE fusion proteins using glutathione-Sepharose. The purified PDE was active [V(max) = 318 +/- 18 nmol min(-1)(mg of protein)(-1)] and inhibited by RO 20-1724, rolipram, and MIX (IC50 values of 2, 0.4, and 40 microM, respectively). The requirement of PDE4D1 for a divalent cation was also examined. It was able to use Mg2+, Co2+, and Mn2+, but not Zn2+, suggesting that it is not a zinc hydrolase as has been proposed for other PDE types. Deletion of both C- and N-terminal regions affected the apparent native size of the enzyme. The C-terminal region was involved in dimer formation, whereas an N-terminal region was responsible for larger aggregates. Removal of the last 35 amino acids of an N-terminal 80-residue highly conserved region (UCR2) resulted in a 6-fold increase in PDE activity, providing evidence that this part of the molecule acts as an intramolecular inhibitor. The availability of a highly purified, enzymatically active protein in substantial quantities has allowed us to directly examine PDE4D1 for the first time.
Kovala
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Department of Biochemistry, University of Western Ontario, London, Canada.
Sanwal
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Ball
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D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D002413:Cations, Divalent / Q000494:pharmacology; D002460:Cell Line; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D007700:Kinetics; D018485:Muscle Fibers, Skeletal / Q000201:enzymology; D009154:Mutation; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D017384:Sequence Deletion; D013329:Structure-Activity Relationship
D002413:Cations, Divalent; D017931:DNA Primers; D011993:Recombinant Fusion Proteins; D015105:3',5'-Cyclic-AMP Phosphodiesterases