Recombinant Expression of a Type IV, cAMP-Specific Phosphodiesterase: Characterization and Structure−Function Studies of Deletion Mutants
| Autor: | B. D. Sanwal, Eric H. Ball, Tom Kovala |
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| Rok vydání: | 1997 |
| Předmět: |
Cations
Divalent Recombinant Fusion Proteins Muscle Fibers Skeletal Gene Expression Biology Biochemistry Cell Line Divalent Structure-Activity Relationship Hydrolase Escherichia coli medicine Animals Cloning Molecular Rolipram DNA Primers Sequence Deletion chemistry.chemical_classification Expression vector Phosphodiesterase Fusion protein Molecular biology Amino acid Kinetics Enzyme chemistry 3' 5'-Cyclic-AMP Phosphodiesterases Mutation Chromatography Gel Electrophoresis Polyacrylamide Gel medicine.drug |
| Zdroj: | Biochemistry. 36:2968-2976 |
| ISSN: | 1520-4995 0006-2960 |
| DOI: | 10.1021/bi9613483 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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