caa a22 4500 397520573 CHVBK 20180308164631.0 cr unu---uuuuu 161202e199503 xx s 000 0 eng 10.1093/jxb/46.3.347 doi (NATIONALLICENCE)oxford-10.1093/jxb/46.3.347 Purification and properties of DNA topoisomerase II from Daucus carota cells [Elektronische Daten] [Daniela Carbonera, Laura Rovati, Fulvio Guano, Alma Balestrazzi] 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 kDNA, requires ATP and Mg2+ for activity. The unknotting activity was sensitive to an inhibitor of the mammalian type II enzyme, the drug VP16 (IC50 32 mmol m−3), whereas inhibitors of DNA gyrase showed a limited effect on activity. The SDS-PAGE analysis of the dsDNA cellulose fraction revealed the presence of four polypeptides of apparent molecular masses of 72, 71, 34, and 33 kDa among which only a polypeptide of about 70 kDa crossreacted with antibodies against yeast topoisomerase II. Immunoprecipitation experiments with monoclonal antibodies to the α and β isoforms of the human enzyme confirmed the recognition of a polypeptide of 70 kDa. The sedimentation coefficient (S) of the topoisomerase II in the phosphocellulose fraction, calculated by analytical glycerol gradient, was 6.1 corresponding to a molecular mass of about 123 kDa. Results suggest the presence in carrot of a protein of molecular mass of 70 kDa having the typical properties of an eukaryotic topoisomerase II and carrying epitopes recognized by MoAbs to both human α and β enzymes. The 70 kDa polypeptide might then represent the monomer of a homodimer enzyme of 123 kDa. © Oxford University Press Research Papers nationallicence Daucus carota nationallicence topoisomerase II nationallicence immunoprecipitation nationallicence Carbonera Daniela Dipartimento di Genetica e Microbiologia ‘A. Buzzati-Traverso', Università di Pavia Via Abbiategrasso 207, I-27100 Pavia, Italy aut Rovati Laura Dipartimento di Genetica e Microbiologia ‘A. Buzzati-Traverso', Università di Pavia Via Abbiategrasso 207, I-27100 Pavia, Italy aut Guano Fulvio Istituto di Genetica Biochimica ed Evoluzionistica del CNR Via Abbiategrasso 207, I-27100 Pavia, Italy aut Balestrazzi Alma Dipartimento di Genetica e Microbiologia ‘A. Buzzati-Traverso', Università di Pavia Via Abbiategrasso 207, I-27100 Pavia, Italy aut Journal of Experimental Botany Oxford University Press 46/3(1995-03), 347-354 0022-0957 46:3<347 1995 46 exbotj https://doi.org/10.1093/jxb/46.3.347 text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1093/jxb/46.3.347 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Carbonera Daniela Dipartimento di Genetica e Microbiologia ‘A. Buzzati-Traverso', Università di Pavia Via Abbiategrasso 207, I-27100 Pavia, Italy aut NATIONALLICENCE 700 1- Rovati Laura Dipartimento di Genetica e Microbiologia ‘A. Buzzati-Traverso', Università di Pavia Via Abbiategrasso 207, I-27100 Pavia, Italy aut NATIONALLICENCE 700 1- Guano Fulvio Istituto di Genetica Biochimica ed Evoluzionistica del CNR Via Abbiategrasso 207, I-27100 Pavia, Italy aut NATIONALLICENCE 700 1- Balestrazzi Alma Dipartimento di Genetica e Microbiologia ‘A. Buzzati-Traverso', Università di Pavia Via Abbiategrasso 207, I-27100 Pavia, Italy aut NATIONALLICENCE 773 0- Journal of Experimental Botany Oxford University Press 46/3(1995-03), 347-354 0022-0957 46:3<347 1995 46 exbotj Metadata rights reserved CC BY-NC-4.0 http://creativecommons.org/licenses/by-nc/4.0 nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-oxford