CAJ | 학술논문

目的：用阴离子交换HPLC分析环核苷酸磷酸二酯酶反应体系。方法：用Shim-Pak WAX-I柱，30 mmol/L的pH 4.0磷酸钾为流动相，洗脱速度0.5 ml/min，254 nm检测，峰面积定量。结果：上述离子交换HPLC体系可分离并定量测定cAMP和AMP。在硫酸铵分级粗制兔肝PDE反应混合物中，cAMP和AMP同杂质完全基线分离；随反应进行，cAMP逐步下降，AMP上升到平台后再下降。用米氏酶的积分速度方程作图分析cAMP下降的进程曲线，发现硫酸铵分级粗制的兔肝PDE的米氏常数为负数。结论：此离子交换HPLC方法可以分析无杂酶干扰时PDE对cAMP的水解。
목적：용음리자교환HPLC분석배핵감산린산이지매반응체계。방법：용Shim-Pak WAX-I주，30 mmol/L적pH 4.0린산갑위류동상，세탈속도0.5 ml/min，254 nm검측，봉면적정량。결과：상술리자교환HPLC체계가분리병정량측정cAMP화AMP。재류산안분급조제토간PDE반응혼합물중，cAMP화AMP동잡질완전기선분리；수반응진행，cAMP축보하강，AMP상승도평태후재하강。용미씨매적적분속도방정작도분석cAMP하강적진정곡선，발현류산안분급조제적토간PDE적미씨상수위부수。결론：차리자교환HPLC방법가이분석무잡매간우시PDE대cAMP적수해。
Objective: Ion-exchange high-performance liquid chromatography (HPLC) was investigated for the analysis of cAMP hydrolysis catalyzed by rabbit liver cyclic nucleotide phosphodiesterase (PDE). Methods: The HPLC column was Shim-Pak WAX-1, mobile phase was 30 mmoI/L potassium phosphate at pH 4.0 eluted at 0.5ml/min, detection was absorbance at 254 nm, and quantification was performed with peak area. Results: cAMP and AMP were well-resolved and quantified by peak area under this HPLC condition. Rabbit liver PDE was partially purified by ammonium sulfate fractionation. In this PDE reaction mixture, residual contaminants after extraction were all baseline-resolved from both cAMP and AMP. The time course of rabbit PDE reaction showed that cAMP continuously dropped but AMP increased to a plateau before dropping. Analysis of cAMP dropping by the integrated Michaels-Menten rate equation of mono-substrate irreversible reaction lead to negative Michaelis constant. Conclusion: This ion-exchange HPLC method was desirable for the analysis of cAMP hydrolysis by PDE without phosphatase interference.