Transfection of COS7 cells with a plasmid encoding the human cyclic AMP-specific PDE4A phosphodiesterase PDE-46 (HSPDE4A4B) led to the expression of a rolipram-inhibited PDE4 activity, which contributed ϳ96% of the total COS cell PDE activity. A fusion protein was generated which encompassed residues (788 -886) at the extreme C terminus of PDE-46 and was used to generate an antiserum that detected PDE-46 in transfected COS7 cells. Immunoblotting studies identified PDE-46 as a ϳ125-kDa species that was associated with both the soluble and particulate fractions. The relative V max of particulate PDE-46 was ϳ56% that of cytosolic PDE-46. Particulate PDE-46 was not solubilized using Triton X-100 or high NaCl concentrations. Immunofluorescence analysis by laser scanning confocal microscopy showed that PDE-46 was located at discrete margins of the cell, indicative of association with membrane cortical regions. The human PDE4A species, h6.1 (HSPDE4A4C), which lacks the N-terminal extension of PDE-46, was found as an entirely soluble species when expressed in COS7 cells. h6.1 was shown to have an ϳ11-fold higher V max relative to that of PDE-46. In doseresponse studies rolipram inhibited particulate PDE-46 at much lower concentrations (IC 50 ‫؍‬ 0.195 M) than those needed to inhibit the cytosolic enzyme (IC 50 ‫؍‬ 1.6 M). The basis of this difference lay in the fact that rolipram served as a simple competitive inhibitor of the cytosol enzyme (K i ‫؍‬ 1.6 M) but as a partial competitive inhibitor of the particulate enzyme (K i ‫؍‬ 0.037 M; K i ‫؍‬ 2.3 M). Particulate PDE-46 thus showed a ϳ60-fold higher affinity for rolipram than cytosolic PDE-46.

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