Badaracco G, Landsberger N, Benfante R
Dipartimento di Genetica e di Biologia dei Microrganismi, Università di Milano, Italy.
Biochem J. 1992 Feb 15;282 ( Pt 1)(Pt 1):249-54. doi: 10.1042/bj2820249.
The ATP-independent type I topoisomerase from the crustacean Artemia franciscana was purified to near-homogeneity. Its activity was measured by an assay that uses the formation of an enzyme-cleaved DNA complex in the presence of the specific inhibitor camptothecin. The purification procedure is reported. Purified topoisomerase is a single-subunit enzyme with a molecular mass of 63 kDa. Immunoblot performed on the different steps of purification shows that the purified 63 kDa peptide is a proteolytic fragment of a protein with a molecular mass of 110 kDa. Similarly to the other purified eukaryotic topoisomerases, the crustacean enzyme does not require a bivalent cation for activity, but is stimulated in the presence of 10 mM-MgCl2; moreover, it can relax both negative and positive superhelical turns. The enzyme activity is strongly inhibited by the antitumour drug camptothecin. The enzyme inhibition is related to the stabilization of the cleavable complex between topoisomerase I and DNA.
对来自卤虫（Artemia franciscana）的不依赖ATP的I型拓扑异构酶进行了纯化，使其接近均一。通过一种检测方法来测定其活性，该方法利用在特异性抑制剂喜树碱存在的情况下形成酶切DNA复合物。报告了纯化过程。纯化后的拓扑异构酶是一种单亚基酶，分子量为63 kDa。对纯化的不同步骤进行免疫印迹分析表明，纯化后的63 kDa肽是一种分子量为110 kDa蛋白质的蛋白水解片段。与其他纯化的真核拓扑异构酶类似，这种甲壳类动物的酶的活性不需要二价阳离子，但在10 mM MgCl2存在的情况下会受到刺激；此外，它可以使负超螺旋和正超螺旋解旋。该酶的活性受到抗肿瘤药物喜树碱的强烈抑制。酶的抑制作用与拓扑异构酶I和DNA之间可裂解复合物的稳定有关。
