Fukata H, Mochida A, Maruyama N, Fukasawa H
Biochemical Laboratory, Kobe Women's University, Hyogo.
J Biochem. 1991 Jan;109(1):127-31. doi: 10.1093/oxfordjournals.jbchem.a123332.
An ATP-independent DNA topoisomerase has been isolated from chloroplasts of cauliflower leaves (Brassica oleracea var. botrytis) through DEAE-cellulose, AF-blue Toyopearl, and hydroxyapatite column chromatography. The sedimentation coefficient and Stokes radius of this enzyme are 3.6S and 3.6 nm, respectively, and the molecular weight of native enzyme is estimated to be 54,000. This enzyme changes the linking number in steps of one. The enzyme activity is stimulated by MgCl2, and this enzyme shows optimum activity at 30 degrees C in the range of 3 mM MgCl2 + 100 mM KCl-10 mM MgCl2 + 50 mM KCl. The enzyme activity was reduced remarkably by N-ethylmaleimide, indicating that a free sulfhydryl group is important for the activity; heparin and ellipticine also reduced the activity. Both cauliflower chloroplast topoisomerase and spinach chloroplast topoisomerase can relax positive supercoils as well as negative supercoils. From these properties, cauliflower chloroplast topoisomerase can be classified as a eukaryotic type I DNA topoisomerase.
一种不依赖ATP的DNA拓扑异构酶已通过二乙氨基乙基纤维素、AF-蓝琼脂糖凝胶和羟基磷灰石柱色谱从花椰菜叶片（甘蓝变种）的叶绿体中分离出来。该酶的沉降系数和斯托克斯半径分别为3.6S和3.6纳米，天然酶的分子量估计为54,000。这种酶以每次一个的步长改变连环数。酶活性受到氯化镁的刺激，并且该酶在30℃、3 mM氯化镁 + 100 mM氯化钾至10 mM氯化镁 + 50 mM氯化钾的范围内表现出最佳活性。N-乙基马来酰亚胺显著降低了酶活性，表明游离巯基对活性很重要；肝素和椭圆玫瑰树碱也降低了活性。花椰菜叶绿体拓扑异构酶和菠菜叶绿体拓扑异构酶都能松弛正超螺旋和负超螺旋。基于这些特性，花椰菜叶绿体拓扑异构酶可归类为真核I型DNA拓扑异构酶。
