Purification and characterization of a type II DNA topoisomerase from bovine calf thymus

B. D. Halligan, K. A. Edwards, Leroy-Fong Liu

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Abstract

We report here the large scale purification of DNA topoisomerase II from calf thymus glands, using the unknotting of naturally knotted P4 phage DNA as an assay for enzymatic activity. Topoisomerase II was purified more than 1300-fold as compared to the whole cell homogenate, with 22% yield. Analysis of the purified enzyme by sodium dodecyl sulfate-polyacrylamide gele electrophoresis revealed two bands of apparent molecular masses of 125 and 140 kDa. Tryptic maps of the two bands indicated that they derive from the same protein. Using these fragments, specific polyclonal antisera to topoisomerase II were raised in rabbits. Immunoblotting of whole cell lysates from various species indicated that topoisomerase II is well conserved among mammals and has a native subunit molecular mass of 180 kDa. Analytical sedimentation and gel filtration were used to determine a sedimentation coefficient of 9.8 S and a Stokes radius of 68 Å. The calculated solution molecular mass of 277 kDa implies a dimer structure in solution. The purified topoisomerase II unknots P4 DNA in an ATP-dependent manner and is highly stimulated in its relaxation activity by ATP. A DNA-stimulated ATPase activity, as has been found with other type II topoisomerases, is associated with the purified enzyme. Approximate kinetic parameters for the ATPase reaction were determined to be: a V(max) of 0.06 nmol of ATP (μg of protein) (min) and K(m) of 0.2 mM in the absence of DNA, and a V(max) of 0.2 nmol of ATP/(μg of protein) (min) and K(m) of 0.4 mM ATP in the presence of supercoiled plasmid DNA.

Original languageEnglish
Pages (from-to)2475-2482
Number of pages8
JournalJournal of Biological Chemistry
Volume260
Issue number4
Publication statusPublished - 1985
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry

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