title: 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-2-oxoquinolyl oxy)] -butyramide (OPC-13135) is one of these derivatives, and the concentration of OPC-13135 producing 50% inhibition of human platelet aggregation induced by 2 micrograms/ml collagen was 5 microM. 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 microM, respectively. We purified over 480-fold the soluble low Km form of cyclic AMP phosphodiesterase from human platelets, using OPC-13135 Sepharose 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 Km value for the substrate cyclic AMP and cyclic GMP of 0.34 and 0.11 microM respectively, and a Vmax value of 85.3 and 19.8 nmole/min/mg protein, respectively. Ki value of the OPC-13135 for the enzyme was 0.015 microM and was of competitive fashion against cyclic AMP.
mesh_terms: D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000037:antagonists & inhibitors / Q000302:isolation & purification*; D001792:Blood Platelets / Q000201:enzymology*; D002846:Chromatography, Affinity; D002847:Chromatography, Agarose; D002850:Chromatography, Gel; D006801:Humans; D007700:Kinetics; D010974:Platelet Aggregation / Q000187:drug effects; D011804:Quinolines / Q000494:pharmacology*; D015363:Quinolones
chemical_list: D011804:Quinolines; D015363:Quinolones; C021294:cilostamide; C042869:OPC 13135; D015105:3',5'-Cyclic-AMP Phosphodiesterases