人脾DNA拓扑异构酶Ⅰ的提纯及其性质的研究  

Purification and Characterization of DNA Topoisomerase Ⅰ From Human Spleen

作　　者：徐桢琦[1] 王理开[1] 徐晓利[1] 

机构地区：[1]中山医科大学生化教研室

出　　处：《生物化学杂志》1990年第4期345-351,共7页

摘　　要：经三个步骤从人脾提纯了拓扑酶Ⅰ。SDS-PAGE和等电聚焦均显示单一蛋白带。分子量77kD,pI7.3。新生霉素和香豆霉素A_1不抑制酶活性。本实验表明,反应最适K^(+)或Na^(+)浓度为180mmol/L。反应活性不要求Mg^(2+),但5mmol/L存在时,活性增加90％。在pH5.0～9.0均有活性,但在7.5～8.0时活性较大。30℃放置24小时或50℃处理30分钟活性基本丧失。最适反应温度为37℃。对-氯汞苯甲酸强烈抑制酶活性。DNA topoisomerase I was purified from human spleen by three steps to give a single band in SDS-PAGE and isoelectric focusing. The molecular weight was 77 kD and the isoelectric point was 7.3. Novobiocin and coumermycin A1 had no effects on the activity. The activity was maximum at 180 mmol/L Na+ or K+, The enzyme maintained its activity in the absence of Mg2+,but in the presence of 5 mmol/L Mg2+, the activity was almost doubled.It could relax super-coiled DNA at pH 5.0-9.0, but the activity was higher at pH7.5-8.0. The optimum reaction temperature was 37℃. Preincubation of the enzyme at 50℃ for 30 min. Or standed at 30℃ for 24 h. resulted in the loss of most activity. The activity was strongly inhibited by p-mercurichlorobenzoic acid, suggesting that sulfhydryl group is essential for its activity.

关 键 词：DNA 拓扑异构酶I 提纯 动力学 

分 类 号：Q558[生物学—生物化学]