THE HUMAN CYCLIC AMP-SPECIFIC PHOSPHODIESTERASE PDE-46 (HSPDE4A4B) EXPRESSED IN TRANSFECTED COS7 CELLS OCCURS AS BOTH PARTICULATE AND CYTOSOLIC SPECIES THAT EXHIBIT DISTINCT KINETICS OF INHIBITION BY THE ANTIDEPRESSANT ROLIPRAM

Transfection of COS7 cells with a plasmid encoding the human cyclic A- specific PDE4A phosphodiesterase PDE-46 (HSPDE4A4B) led to the express ion of a rolipram-inhibited PDE4 activity, which contributed similar t o-96% of the total COS cell PDE activity, A fusion protein was generat ed which encompassed residues (788-886) at the extreme C terminus of P DE-46 and was used to generate an antiserum that detected PDE-46 in tr ansfected COS7 cells, Immunoblotting studies identified PDE-46 as a si milar to 125-kDa species that was associated with both the soluble and particulate fractions. The relative V-max of particulate PDE-46 was s imilar to 56% that of cytosolic PDE-46. Particulate PDE-46 was not sol ubilized using Triton X-100 or high NaCl concentrations, Immunofluores cence analysis by laser scanning confocal micros copy showed that PDE- 46 was located at discrete margins of the cen, indicative of associati on with membrane cortical regions. The human PDE4A species, h6.1 (HSPD E4A4C), which lacks the N-terminal extension of PDE-46, was found as a n entirely soluble species when expressed in COS7 cells, h6.1 was show n to have an similar to 11-fold higher V-max relative to that of PDE-4 6. In dose-response studies rolipram inhibited particulate PDE-46 at m uch lower concentrations (IC50 = 0.195 mu M) than those needed to inhi bit the cytosolic enzyme (IC50 = 1.6 mu M). The basis of this differen ce lag in the fact that rolipram served as a simple competitive inhibi tor of the cytosol enzyme (K-i = 1.6 mu M) but as a partial competitiv e inhibitor of the particulate enzyme (K-i = 0.037 mu M; K-i' = 2.3 mu M) Particulate PDE-46 thus showed a similar to 60-fold higher affinit y for rolipram than cytosolic PDE-46.