Intact cell and cell-free phosphorylation and concomitant activation of a low Km, cAMP phosphodiesterase found in human platelets.
A monoclonal antibody (CGI-5) directed against the cGMP-inhibited phosphodiesterase isolated from bovine heart was used to examine the phosphorylation of this isozyme in human platelets. PGE1 promoted the phosphorylation of this isozyme, identified as a 110 kDa peptide following SDS-gel electrophoresis. Phosphorylation resulted in approximately a 40% increase in the cGMP-inhibited phosphodiesterase activity. Cell-free experiments demonstrated that cAMP-dependent protein kinase phosphorylated the cGMP-inhibited phosphodiesterase, and that this could be blocked by the heat stable inhibitor peptide (PKI). Phosphorylation of the cGMP-inhibited phosphodiesterase increases the Vmax for cAMP hydrolysis approximately 50%, but does not affect the Km for cAMP (0.12 microM).
Macphee
C H
CH
Department of Pharmacology, University of Washington, Seattle.
Reifsnyder
D H
DH
Moore
T A
TA
Beavo
J A
JA
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000037:antagonists & inhibitors / Q000097:blood*; D000527:Alprostadil / Q000494:pharmacology; D001792:Blood Platelets / Q000201:enzymology*; D000242:Cyclic AMP / Q000494:pharmacology; D006152:Cyclic GMP / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D006801:Humans; D007163:Immunosorbent Techniques; D007527:Isoenzymes / Q000097:blood*; D007700:Kinetics; D010766:Phosphorylation; D011494:Protein Kinases / Q000097:blood
D007527:Isoenzymes; D000242:Cyclic AMP; D011494:Protein Kinases; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D000527:Alprostadil; D006152:Cyclic GMP