Purification of cyclic adenosine monophosphate phosphodiesterase from human platelets using new-inhibitor sepharose chromatography

Abstract

Cilostamide derivatives are potent inhibitors of human platelet aggregation and selectively inhibit human platelet cyclic adenosine monophosphate (cyclic AMP) phosphodiesterase. N-Cyclohexyl- N-(2-hydroxybutyl)-5-[6-1,2,3,4-tetrahydro-2oxoquinolyloxy)]-butyramide (OPC-13135) is one of these derivatives, and the concentration of OPC-13135 producing 50% inhibition of human platelet aggregation induced by 2 μg/ml collagen was 5 μM. On the other hand, the concentrations of OPC-13135 producing 50% inhibition of human platelet cyclic AMP phosphodiesterase and cyclic guanosine monophosphate (cyclic GMP) phosphodiesterase were 0.073 and 21.8 μM, respectively. We purified over 480-fold the soluble low K m form of cyclic AMP phosphodiesterase from human platelets, using OPC-13135 Sehparose column as a final step in the purification procedure. The purified protein has a molecular weight of 175,000, determined by gel filtration and is an acidic protein, as determined by isoelectric focussing (pI = 4.9). Kinetic measurements indicated that the enzyme protein had a K m , value for the substrate cyclic AMP and cyclic GMP of 0.34 and 0.11 μM respectively, and a V max value of 85.3 and 19.8 nmole/min/mg protein, respectively. K i value of the OPC-13135 for the enzyme was 0.015 μM and was of competitive fashion against cyclic AMP.