Certain inotropic agents act by specifically inhibiting human myocardial cG1 PDE, some of which is associated with the sarcoplasmic reticulum (SR). Human cardiac CGI PDE CDNA, which encodes a protein (subunit Mr, - 125 Kda), with predicted N-terminal region-membrane association domains and a C-terminal region-catalytic domain (conserved among all mammalian PDEs) separated by a putative regulatory domain, was expressed in Sf9 cells infected with a recombinant CGI PDE Baculovirus transfer vector (pVL1393, PharMingen). CGI PDE, found predominantly in association with Sf9 cell particulate fractions, was sensitive inhibition by CGMP and OPC3869 (cilostamide), but not rolipram. The recombinant CGI PDE and human cardiac microsomal CGI PDE exhibited similar kinetic properties with Km values of 0.14 to 0.19 microM. A partial cardiac CDNA clone (n.2), encoding a truncated (54-Kda) CGI PDE containing the conserved catalytic domain, was recovered predominantly in Sf9 cell cytosol. This may be due to the absence of membrane-association domains in the truncated CGI PDE. The recombinant particulate CGI PDE could be released by the detergent C13E12 or salt alone and to somewhat greater extent by combination of salt and detergent. These results suggest that Baculovirus expression systems will help understand mechanisms of CGI PDE association with human myocardial SR.
