离子交换高效液相色谱法分析兔肝环核苷酸磷酸二酯酶反应体系  

Lon-exchange high-performance liquid chromatography analysis of rabbit liver cyclic nucleotide phosphodiesterase reaction mixture

作　　者：廖飞[1] 张晓萍[2] 周岐新[3] 杨俊清[3] 曾昭淳[1] 陈文缘[1] 汤先觉[4] 

机构地区：[1]重庆医科大学基础医学院生物化学教研室 [2]重庆医科大学中心实验室 [3]重庆医科大学基础医学院药理学教研室 [4]重庆医科大学基础医学院化学教研室,重庆400016

出　　处：《重庆医科大学学报》2001年第1期20-22,共3页Journal of Chongqing Medical University

摘　　要：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-1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。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 mmol/L potassium phosphate at pH 4.0 eluted at 0.5 ml/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.

关 键 词：环核苷酸磷酸二酯酶 离子交换高效液相色谱法 3 5-cAMP 5-AMP 

分 类 号：Q556.103[生物学—生物化学]