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Cyclic AMP phosphodiesterase (PDE) is a regulatory enzyme in human platelets. Inhibitors of this enzyme raise intracellular cAMP which prevents platelet activation. Little is known about the biochemistry of this enzyme. PDE was isolated from human platelet concentrates by nitrogen bomb cavitation. The specific activity of PDE in cell lysate was 0.064 nmoles cAMP hydrolysed/min/mg protein at 22° , 1 µM cAMP. Eighty percent of the activity appeared in the 100,000 × g supernatant fraction. Chromatography was performed on DEAE cellulose equibrated with 50 mM Tris- acetate pH 6.0, 3.75 mM 2-mercaptoethanol. A linear gradient with a limiting salt concentration of 1.0 M Na acetate separated two peaks of PDE activity. The first had a for cAMP of &gt;100 µM; the second had a Km for cAMP of 5 µM. The lower enzyme was further purified by adsorption on blue dextran Sepharose in 50 mM Tris pH 7.5, 2 mM MgCl2 followed by affinity elution with 1 mM cAMP in the same buffer. These steps resulted in a 1700 purification of the enzyme (113 nmoles/min/mg). The compound 2’-0-iodohydrin- p-cAMP (IH-cAMP) is a cAMP derivative with an alkylating side chain. Incubation of PDE with 5 mM IH-cAMP at 37° resulted in 88% inactivation of the enzyme at 15 hours, compared to a control, with a corrected pseudo-first-order rate constant of 0.144 h-1. When cAMP (100 µM) was included in the inactivation mixture the corrected pseudo-first-order rate constant decreased to 0.064 h-1l. Thus, a 20-fold excess of cAMP protected 56% against inactivation by IH- cAMP. The inhibition was not reversed by gel filtration of the inactivated enzyme which removed IH-cAMP. These results suggest that IH-cAMP reacts with the active site of PDE to irreversibly inactivate the enzyme. IH-cAMP should prove to be a useful tool in understanding the chemistry of the active site of this important enzyme.

Inactivation Of Purified Human Platelet Cyclic Amp Phosphodiesterase By The Affinity Label 2’-O-Iodohydrin-P-Cyclic Amp