真核细胞顺乌头酸酶活性检测新技术——胶内酶活性分析法

In-Gel Activity Assay: A Novel Method for Eukaryotic Aconitase Activity Assay

顺乌头酸酶(aconitase,Aco)是细胞内重要的铁硫蛋白酶,它催化细胞内柠檬酸经中间产物顺乌头酸生成异柠檬酸.真核细胞中顺乌头酸酶有两种,分别定位在细胞质的顺乌头酸酶1(cAco)和定位在线粒体的顺乌头酸酶2(m-Aco).检测它们活性的变化能敏感地反映出细胞中能量代谢、自由基产生、铁硫簇组装及铁代谢水平的改变.顺乌头酸酶活性的传统检测方法通常是测定细胞中总的顺乌头酸酶活性,该方法难以准确区分出c-Aco和m-Aco各自的活性变化.因此我们建立一种胶内酶活性分析法检测顺乌头酸酶活性.该方法利用非变性电泳技术将c-Aco和m-Aco浓缩分离,通过泡染底物显色,条带颜色深浅反映了酶活性的强弱.同时,比较了胶内酶活性分析法和分光光度法检测细胞内c-Aco和m-Aco的活性,并对比检测了过氧化氢处理细胞前后Aco活性的变化.结果显示,这两种方法均可敏感地检测出Aco的活性改变,并有广泛的细胞系实用性,但胶内酶活性分析法可区别测定c-Aco和m-Aco活性,不需繁琐的细胞质和线粒体分离,简便易行.文中介绍的线粒体分离纯化技术也为线粒体功能深入研究提供了一个快速、高效的分离纯化方法.

Aconitases are important Fe-S proteins which can catalyze the reaction from citric acid to isocitrate via the intermediate cis-aconitate. In eukaryotic cells,there are two isoenzymes of aconitases,which are respectively located in the cytoplasm（ aconitase 1,c-Aco） and mitochondria（ aconitase 2,mAco）. The activity changes of these two aconitases can sensitively reflect the levels of energy metabolism,free radical generation,iron sulfur cluster assembly and the homeostasis of iron metabolism. Total cell lysates were used to detect the activity of aconitase in traditional methods, it obviously does not respectively reflect the activity change of c-Aco and m-Aco. In this paper,we set up a novel method for eukaryotic aconitase activity assay. The c-Aco and m-Aco were concentrated and separated by the nondenaturation electrophoresis. After electrophoresis,the gel was incubated in the substrate buffer until visible bands appeared,quantitation was performed using Bio-Rad Image Software. And then,in-gelactivity assay and spectrophotometry were used to detect the activity of m-Aco and c-Aco in several cell lines. Otherwise,two assays were used to monitor the activity changes of m-Aco and c-Aco after treatment with gradient hydrogen peroxide. The results demonstrated that these two assays can widely be used to detect the activity of m-Aco and c-Aco in different cell lines or fresh specimens. And the in-gel activity assay costs less time and avoids separating mitochondria from the cell lysate. In addition,nonionic detergent was used to isolate mitochondria in this work,and it provided a quick and efficient method for rapid mitochondrial isolation.