Expression, purification, and characterization of human cAMP-specific phosphodiesterase (PDE4) subtypes A, B, C, and D.
Although four members (A, B, C, and D) of the cAMP-specific phosphodiesterase (PDE4) family have been cloned by different groups, no study comparing the characteristics of purified human PDE4 subtypes has been published. In this study, we have expressed human PDE4 A, B, C, and D in insect (SF9) cells by using the baculovirus expression system, purified the expressed proteins, and compared their characteristics. The recombinant PDE4 subtypes all showed catalytic activity for cAMP with a K(m) of 1-5 microM. V(max) values differed significantly among these subtypes with the following order: C > B > A > D. PDE4 A, B, C, and D showed a very similar Mg2+ dependence profile. PDE4 B and C showed similar pH profiles with the optimal pH being 8.0. The pH profiles of PDE4 A and D were very different from each other and from those of B and C, with the optimal pH being 6.5 and 7.5, respectively. Furthermore, although PDE4 A, B, C, and D were all inhibited by the standard PDE4 inhibitors rolipram, Ro20-1724, and etazolate, the inhibitory potency varied. Thus, by several criteria including kinetics, pH dependency, and inhibitor sensitivity, various PDE4 subtypes differ significantly from one another.
Wang
P
P
Allergy Department, Schering-Plough Research Institute, Kenilworth, New Jersey 07033, USA.
Myers
J G
JG
Wu
P
P
Cheewatrakoolpong
B
B
Egan
R W
RW
Billah
M M
MM
D015105:3',5'-Cyclic-AMP Phosphodiesterases; D012368:4-(3-Butoxy-4-methoxybenzyl)-2-imidazolidinone / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D054703:Cyclic Nucleotide Phosphodiesterases, Type 4; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D004974:Etazolate / Q000494:pharmacology; D015870:Gene Expression; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D008969:Molecular Sequence Data; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D010727:Phosphoric Diester Hydrolases / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification*; D011760:Pyrrolidinones / Q000494:pharmacology; D020889:Rolipram; D018411:Spodoptera
D017931:DNA Primers; D018076:DNA, Complementary; D010726:Phosphodiesterase Inhibitors; D011760:Pyrrolidinones; D012368:4-(3-Butoxy-4-methoxybenzyl)-2-imidazolidinone; D010727:Phosphoric Diester Hydrolases; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D054703:Cyclic Nucleotide Phosphodiesterases, Type 4; D008274:Magnesium; D004974:Etazolate; D020889:Rolipram