Mukherjee S K, Reddy M K, Kumar D, Tewari K K
International Centre for Genetic Engineering and Biotechnology, New Delhi, India.
J Biol Chem. 1994 Feb 4;269(5):3793-801.
A 69-kDa protein with topoisomerase I activity has been homogeneously purified from the chloroplasts of pea leaves. The topoisomerase properties are detected in crude lysate of pea chloroplasts using the technique of transferring 32P radioactivity from the 32P-labeled DNA to the protein. The purified enzyme relaxes both positive and negative supercoils in topological steps of unity without requiring magnesium ions. The enzyme is sensitive to topoisomerase I-specific inhibitors like camptothecin and berenil, and unaffected by reagents like novobiocin and doxorubicin at the topoisomerase II-inhibitory dosage. In the presence of the enzyme, supercoiled DNA is nicked, and the 3'-phosphoryl end of the nick becomes covalently linked with the enzyme. A tyrosine residue of the enzyme is responsible for the covalent linkage. Rabbit antiserum raised against the 16-mer peptide spanning the active residues of human topoisomerase I recognizes the 69-kDa protein within the crude lysate of pea chloroplasts as does the antiserum to the purified 69-kDa protein. From the enzymatic characteristics, the protein has been classified as a eukaryotic type I topoisomerase.
一种具有拓扑异构酶I活性的69 kDa蛋白质已从豌豆叶片的叶绿体中被均匀纯化。利用将32P放射性从32P标记的DNA转移到蛋白质上的技术，在豌豆叶绿体的粗裂解物中检测到了拓扑异构酶的特性。纯化后的酶无需镁离子，以单位拓扑步长松弛正超螺旋和负超螺旋。该酶对拓扑异构酶I特异性抑制剂如喜树碱和贝尼尔敏感，在拓扑异构酶II抑制剂量下不受新生霉素和阿霉素等试剂的影响。在该酶存在的情况下，超螺旋DNA被切开，切口的3'-磷酸末端与酶形成共价连接。该酶的一个酪氨酸残基负责共价连接。针对跨越人类拓扑异构酶I活性残基的16聚体肽产生的兔抗血清，与针对纯化的69 kDa蛋白质的抗血清一样，能识别豌豆叶绿体粗裂解物中的69 kDa蛋白质。根据酶学特性，该蛋白质已被归类为真核I型拓扑异构酶。
