CHARACTERIZATION OF 2 HUMAN CAMP-SPECIFIC PHOSPHODIESTERASE SUBTYPES EXPRESSED IN BACULOVIRUS-INFECTED INSECT CELLS

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 (similar to 3 mu M) for cAMP (with no cGMP hydrolyzing activity) and were inhibited by R-rolipram with apparent K(i)s of 0.38 and 0.25 mu M, respectively. The recombinant enzymes also contained saturable, stereoselective and high-affinity rolipram-binding sites (K-d similar to 2 nM). Thus, insect cell-derived hPDE4s possess kinetic properties analogous to native enzymes as well as to recombinant enzymes produced in yeast.