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Characterization of two human cAMP‐specific phosphodiesterase subtypes expressed in baculovirus‐infected insect cells.
Author(s) -
Amegadzie Bernard Y.,
Hanning Charles R.,
McLaughlin Megan M.,
Burman Miriam,
Cieslinski Lenora B.,
Livi George P.,
Torphy Theodore J.
Publication year - 1995
Publication title -
cell biology international
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.932
H-Index - 77
eISSN - 1095-8355
pISSN - 1065-6995
DOI - 10.1006/cbir.1995.1091
Subject(s) - recombinant dna , rolipram , spodoptera , phosphodiesterase , baculoviridae , sf9 , enzyme , microbiology and biotechnology , biology , biochemistry , yeast , gene
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 K m (∼ 3 μM) for cAMP (with no cGMP hydrolyzing activity) and were inhibited by R‐rolipram with apparent K i s of 0.38 and 0.25 μM, respectively. The recombinant enzymes also contained saturable, stereoselective and high‐affinity rolipram‐binding sites (K d ∼ 2 nM). Thus, insect cell‐derived hPDE4s possess kinetic properties analogous to native enzymes as well as to recombinant enzymes produced in yeast.