Purification and characterization of DNA topoisomerase II from XXXX thymus associated with polypeptides of 175 and 150 kDa.

@entity1025 @entity3281 @entity1219 @entity17699 @entity653 @entity692 @entity2975 @entity1329 @entity291 @entity855
DNA topoisomerase II was purified from @entity291 thymus nuclei by a simple and fast four-step procedure: selective @entity1329 precipitation, chromatography on blue-Sepharose and @entity692 , followed by ultracentrifugation on a @entity653 gradient. Starting from 300 g thymus glands, this procedure yields 0.7 mg of homogeneous topoisomerase II. The final product is free of any nucleolytic, proteolytic or topoisomerase I activity. Dodecylsulfate/ @entity2975 gel electrophoresis reveals two bands with apparent molecular masses of 175 and 150 kDa. Analytical gel filtration and sedimentation on isokinetic sucrose gradients were used to determine the Stokes' radius as 6.4 nm and the sedimentation coefficient as 9.5 S, indicating a dimeric structure for the native enzyme. The purified topoisomerase II is strictly dependent on @entity855 or @entity17699 , the Km values of which were 0.14 mM and 0.5 mM, respectively. @entity1025 is an essential cofactor for the reaction at concentrations between 0.5-8 mM, with an optimum at 4 mM. @entity1025 can be substituted by @entity3281 at concentrations between 0.2-0.4 mM. Both the relaxation and the catenation reaction exhibit a salt optimum at 130 mM @entity1219 . At concentrations below 30 mM and above 200 mM, the enzyme is inactive. The pH is optimal between 8 and 9.5 using Tris buffers.
