Characterization of two human cAMP-specific phosphodiesterase subtypes expressed in baculovirus-infected insect cells
| Autor: | Charles R. Hanning, Megan M. McLaughlin, George P. Livi, Bernard Y. Amegadzie, L. B. Cieslinski, Miriam Burman, Theodore J. Torphy |
|---|---|
| Rok vydání: | 1995 |
| Předmět: |
Molecular Sequence Data
Spodoptera law.invention law Animals Humans Amino Acid Sequence Cells Cultured chemistry.chemical_classification Antiserum Insect cell Base Sequence biology Phosphodiesterase Cell Biology General Medicine biology.organism_classification Molecular biology Recombinant Proteins Yeast Enzyme Activation Isoenzymes Kinetics Enzyme chemistry Biochemistry 3' 5'-Cyclic-AMP Phosphodiesterases Recombinant DNA Baculoviridae |
| Zdroj: | Cell Biology International. 19:477-484 |
| ISSN: | 1065-6995 |
| DOI: | 10.1006/cbir.1995.1091 |
| Popis: | Recombinant baculoviruses were constructed to express cDNAs encoding two distinct subtypes of human cAMP-specific phosphodiesterase (hPDE4A and hPDE4B). Infection of Spodoptera frugiperda insect cells with the appropriate recombinant baculoviruses resulted in high level production of biologically-active protein as measured by enzymatic activity and immunoblotting using subtype-specific anti-hPDE4 antisera. Both recombinant proteins showed catalytic activity with a low Km (approximately 3 microM) for cAMP (with no cGMP hydrolyzing activity) and were inhibited by R-rolipram with apparent Kis of 0.38 and 0.25 microM, respectively. The recombinant enzymes also contained saturable, stereoselective and high-affinity rolipram-binding sites (Kd approximately 2 nM). Thus, insect cell-derived hPDE4s possess kinetic properties analogous to native enzymes as well as to recombinant enzymes produced in yeast. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|
Plný text