PURIFICATION AND PROPERTIES OF DNA TOPOISOMERASE-II FROM DAUCUS-CAROTA CELLS

Topoisomerase II was partially purified from Daucus carota cells by a procedure including ammonium sulphate fractionation, ion-exchange, and affinity chromatography steps, The type II enzyme, identified for its ability to unknot knotted P4 DNA and decatenate Trypanosoma cruzi kDN A, requires ATP and Mg2+ for activity. The unknotting activity was sen sitive to an inhibitor of the mammalian type II enzyme, the drug VP16 (IC50 32 mmol m(-3)), whereas inhibitors of DNA gyrase showed a limite d effect on activity. The SDS-PAGE analysis of the dsDNA cellulose fra ction revealed the presence of four polypeptides of apparent molecular masses of 72, 71, 34, and 33 kDa among which only a polypeptide of ab out 70 kDa crossreacted with antibodies against yeast topoisomerase II . Immunoprecipitation experiments with monoclonal antibodies to the al pha and beta isoforms of the human enzyme confirmed the recognition of a polypeptide of 70 kDa. The sedimentation coefficient (S) of the top oisomerase II in the phosphocellulose fraction, calculated by analytic al glycerol gradient, was 6.1 corresponding to a molecular mass of abo ut 123 kDa, Results suggest the presence in carrot of a protein of mol ecular mess of 70 kDa having the typical properties of an eukaryotic t opoisomerase II and carrying epitopes recognized by MoAbs to both huma n alpha and beta enzymes. The 70 kDa polypeptide might then represent the monomer of a homodimer enzyme of 123 kDa.