Fukata H, Fukasawa H
J Biochem. 1982 Apr;91(4):1337-42. doi: 10.1093/oxfordjournals.jbchem.a133820.
DNA topoisomerases which remove superhelical turns in closed circular DNA have been isolated from cauliflower inflorescences using polyethylene glycol fractionation, ammonium sulfate precipitation, and column chromatography on CM-Sephadex or CM-cellulose and DNA-cellulose. Two distinct enzymes, topoisomerase-I and ATP-dependent topoisomerase, were separated clearly by CM-Sephadex or CM-cellulose, and partially characterized using agarose gel electrophoresis with plasmid pBR322 DNA. Topoisomerase-I acts like other eucaryotic DNA topoisomerases in the absence of ATP, is stimulated by spermidine and inhibited by EDTA. The ATP-dependent topoisomerase acts like topoisomerase-I only in the presence of ATP in the reaction medium, is inhibited by spermidine and EDTA, and does not introduce supertwists into closed duplex DNA or produce catenate aggregates under the present reaction conditions.
利用聚乙二醇分级分离、硫酸铵沉淀以及在CM - 葡聚糖凝胶或CM - 纤维素和DNA - 纤维素上进行柱色谱法，从菜花的花序中分离出了能去除闭环DNA中超螺旋结构的DNA拓扑异构酶。通过CM - 葡聚糖凝胶或CM - 纤维素能清晰地分离出两种不同的酶，即拓扑异构酶I和ATP依赖型拓扑异构酶，并使用琼脂糖凝胶电泳结合质粒pBR322 DNA对其进行了部分特性分析。拓扑异构酶I在无ATP时的作用类似于其他真核生物DNA拓扑异构酶，可被亚精胺激活，被EDTA抑制。ATP依赖型拓扑异构酶仅在反应介质中有ATP时的作用类似于拓扑异构酶I，可被亚精胺和EDTA抑制，并且在当前反应条件下不会将超螺旋引入闭环双链DNA或产生连环聚集体。
