Characterization of two 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 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.
Amegadzie
B Y
BY
Department of Gene Expression Sciences, SmithKline Beecham Pharmaceuticals, King of Prussia, PA 19406, USA.
Hanning
C R
CR
McLaughlin
M M
MM
Burman
M
M
Cieslinski
L B
LB
Livi
G P
GP
Torphy
T J
TJ
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D001483:Base Sequence; D002478:Cells, Cultured; D004789:Enzyme Activation; D006801:Humans; D007527:Isoenzymes / Q000096:biosynthesis* / Q000235:genetics; D007700:Kinetics; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D018411:Spodoptera / Q000201:enzymology / Q000821:virology
D007527:Isoenzymes; D011994:Recombinant Proteins; D015105:3',5'-Cyclic-AMP Phosphodiesterases