离子交换高效液相层析法测定腺苷酸环化酶活性  

Assay of Adenylyl Cyclase Activity by Ion-exchange High-performance Liquid Chromatography

作　　者：廖飞[1] 张晓萍[2] 周岐新[3] 曾昭淳[1] 陈文缘 

机构地区：[1]重庆医科大学生物化学教研室,重庆400016 [2]重庆医科大学生物化学教研室中心实验室,重庆400016 [3]重庆医科大学生物化学教研室药理教研室,重庆400016

出　　处：《生物化学与生物物理学报》2000年第6期661-664,共4页

摘　　要：用离子交换高效液相层析 (HPLC)分离并同步测定脑组织腺苷酸环化酶 (AC)反应生成的cAMP ,从而直接测定AC活性。10 0℃加热 1min终止AC反应后 ,混合物在 0℃放置 2h。在此期间 ,反应体系中残留的ATP水解酶将反应混合物中大部分剩余的ATP水解除去。用二氯甲烷抽提除去罂粟碱等干扰物质 ,使混合物中的cAMP同其余物质在Shim PakWAX 1阴离子交换HPLC柱上基线分离 ,并可定量测定。用此方法发现 ,10min内cAMP基本线性生成 ,且反应 10min生成的cAMP量同酶蛋白量成正比。用此法测得小鼠脑组织腺苷酸环化酶活性为每克蛋白质 4 2nmol/min。此结果与经典同位素法测得的结果接近。此离子交换HPLC法测定AC活性比经典同位素法更简便。Adenylyl cyclase(AC)activity was determined by directly quan ti fying the product cAMP with ion exchange HPLC. When AC reaction was terminated , the reaction mixture was kept at 0 ℃ for 2 h to remove residual ATP utilizing the action of ATP hydrolyzing enzymes existed in the crude AC preparation of m embrane pellet. Papaverine was removed by the dichloromethane extraction, and c AMP was quantified by chromatography on a WAX 1 HPLC column with baseline reso lution. It was found that cAMP was linearly formed within 10 min of the reaction , and the amount of cAMP formed was proportional to the amount of enzyme. Mouse brain AC activity determined by this method was 42 nmol/min per gram of protein, similar to that obtained by the classical labeled ATP method . Thi s ion exchange HPLC was more rapid and convenient.

关 键 词：腺苷酸环化酶 离子交换高效液相层析 活性测定 

分 类 号：Q503[生物学—生物化学]