Partial purification and characterization of type I DNA topoisomerase from Bacillus stearothermophilus.
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Type I DNA topoisomerase was partially purified from Bacillus stearothermophilus by ammonium sulfate precipitation and column chromatographies on phospho-cellulose, DEAE-cellulose and heparin-agarose. On heparin-agarose chromatography, topoisomerase I activity was separated into three fractions (designated Fractions A, B, and C). Each fraction was further subjected to gel filtration on Sephacryl S-200. From electrophoretic analysis on polyacrylamide gel, Fraction A was found to contain two enzyme species having molecular weights of 110, 000 and 100, 000, and Fraction B one enzyme species with a molecular weight of 80, 000. The molecular weight of the enzyme in Fraction C was estimated to be around 150, 000 by gel filtration. The enzymes in Fractions A and B exhibited little activity in the presence of Mg2+, while the activity was increased remarkably by NaCI with Mg2+. No activity was observed in the presence of NaCI alone. The enzyme in Fraction C required only Mg2+ for full activity. With Fraction A, the topoisomerase Iinduced cleavage sites on tetracycline-resistant plasmid pNSI (2.55 megadaltons) were mapped. Fraction A cleaved the DNA at ten specific sites. These sites were compared to those of the Haemophilus gallinarum enzyme, which have already been mapped (Shishido et al. (1983) Biochim. Bioplzys. Acta 740, 108). The results showed that there is a remarkable coincidence between the cleavage sites induced by the B. stearothermophilus and H. gallinarurn enzymes.
- 社団法人 日本生化学会の論文
社団法人 日本生化学会 | 論文
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