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| 1984/11/01 |
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Purification of cyclic adenosine monophosphate phosphodiesterase from human platelets using new-inhibitor Sepharose chromatography.
Biochem Pharmacol. 1984 Nov 1;33(21):3339-44.
Purification of cyclic adenosine monophosphate phosphodiesterase from human platelets using new-inhibitor Sepharose chromatography.
Umekawa H, Tanaka T, Kimura Y, Hidaka H.
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.
PMID: 6093810 [PubMed - indexed for MEDLINE]
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