Isolation and characterization of DNA topoisomerase II from cauliflower inflorescences

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Abstract
Type II DNA topoisomerase has been isolated from inflorescences of cauliflower (Brassica oleracea var. botrytis) through a sequence of polyethylene glycol fractionation, ammonium sulfate precipitation, and column chromatography on CM-Sephadex, hydroxyapatite and phosphocellulose. The molecular weight of the native enzyme, based on sedimentation coefficient (9S) and gel filtration analysis (Stokes radius, 60 Å), was estimated to be 223 000. This enzyme was able to catalyze fully the relaxation of supercoiled DNA by breaking and then rejoining the double-stranded DNA. The breaking reaction was reversible by a change in salt concentrations. When an antitumor drug, 4′-(9-acridinylamino)-methanesulfon-m-anisidide, was added to the topoisomerase reaction, DNA cleavage fragments were accumulated; and this suggested that the drug interfered with the reaction at the rejoining step. This enzyme also catalyzed the formation of DNA catenanes in the presence of 8% polyethylene glycol or histone H1, while few catenanes were formed in the presence of spermidine, which was highly effective on a bacterial enzyme.