By. Amegadzie et al., CHARACTERIZATION OF 2 HUMAN CAMP-SPECIFIC PHOSPHODIESTERASE SUBTYPES EXPRESSED IN BACULOVIRUS-INFECTED INSECT CELLS, Cell biology international, 19(6), 1995, pp. 477-484
Recombinant baculoviruses were constructed to express cDNAs encoding t
wo 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 productio
n of biologically-active protein as measured by enzymatic activity and
immunoblotting using subtype-specific anti-hPDE4 antisera. Both recom
binant 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 an
d high-affinity rolipram-binding sites (K-d similar to 2 nM). Thus, in
sect cell-derived hPDE4s possess kinetic properties analogous to nativ
e enzymes as well as to recombinant enzymes produced in yeast.