EXPRESSION, PURIFICATION, AND CHARACTERIZATION OF HUMAN CAMP-SPECIFICPHOSPHODIESTERASE (PDE4) SUBTYPE-A,SUBTYPE-B, SUBTYPE-C, AND SUBTYPE-D

Although four members (A, B, C, and D) of the cAMP-specific phosphodie sterase (PDE4) family have been cloned by different groups, no study c omparing 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, puri fied the expressed proteins, and compared their characteristics. The r ecombinant PDE4 subtypes all showed catalytic activity for cAMP with a K-m of 1-5 mu M. V-max values differed significantly among these subt ypes with the following order: C > B > A > D. PDE4 A, B, C, and D show ed 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, w ith the optimal pH being 6.5 and 7.5, respectively. Furthermore, altho ugh PDE4 A, B, C, and D were all inhibited by the standard PDE4 inhibi tors rolipram, Ro20-1724, and etazolate, the inhibitory potency varied . Thus, by several criteria including kinetics, pH dependency, and inh ibitor sensitivity, various PDE4 subtypes differ significantly from on e another. (C) 1997 Academic Press.