Purification and characterization of a type II DNA topoisomerase from bovine calf thymus.
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 gel 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 A. 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 Vmax of 0.06 nmol of ATP/(micrograms of protein) (min) and Km of 0.2 mM in the absence of DNA, and a Vmax of 0.2 nmol of ATP/(micrograms of protein) (min) and Km of 0.4 mM ATP in the presence of supercoiled plasmid DNA.
Halligan
B D
BD
Edwards
K A
KA
Liu
L F
LF
D000255:Adenosine Triphosphate / Q000378:metabolism / Q000494:pharmacology; D000818:Animals; D001435:Bacteriophages; D002417:Cattle; D002467:Cell Nucleus / Q000201:enzymology; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002845:Chromatography; D004250:DNA Topoisomerases, Type II / Q000302:isolation & purification* / Q000378:metabolism; D004279:DNA, Viral / Q000378:metabolism; D005590:Fractional Precipitation; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D013950:Thymus Gland / Q000201:enzymology*
D004279:DNA, Viral; D046911:Macromolecular Substances; D000255:Adenosine Triphosphate; D004250:DNA Topoisomerases, Type II