Characterization of topoisomerase I and II activities in nuclear extracts during callogenesis in immature embryos of Zea mays.
We have characterized the topoisomerase I and II activities in nuclear extracts from immature embryos of Zea mays and the effect of the treatment with 2,4-dichlorophenoxyacetic acid (2,4-D) and abscisic acid (ABA). These extracts were shown to be essentially devoid of protease and nuclease activities and they were tested for their ability to relax supercoiled DNA, unknotting P4 DNA and catenate circular duplex DNA under catalytic conditions. Unknotting and catenation reactions are strictly magnesium- and ATP-dependent, but not the relaxation of circular supercoiled DNA allowing the detection of both topoisomerase I and II activities. Two cytotoxic drugs, camptothecin, a plant alkaloid that inhibits eukaryotic topoisomerase I, and epipodophyllotoxin VM-26 (teniposide) that inhibits topoisomerase II, have been assayed in our extracts showing similar inhibitory effects on topoisomerase enzymes. Alkaline phosphatase treatment of nuclear extracts abolishes both topoisomerase activities. Nuclear extracts from embryos treated with 2,4-D showed 200% increase on topoisomerase II activity as compared with untreated ones, but only residual activity was detected in ABA-treated embryos. Nuclear extracts from hormone-treated and untreated embryos showed similar topoisomerase I activity with deviations of less than 25%. These differences are discussed in terms of possible post-translational modifications of the enzymes associated with the increase in proliferation activity of calli.
Carballo
M
M
Departamento de Genética Molecular, Centro de Investigación y Desarrollo, CSIC, Barcelona, Spain.
Giné
R
R
Santos
M
M
Puigdomènech
P
P
D015084:2,4-Dichlorophenoxyacetic Acid / Q000494:pharmacology; D000040:Abscisic Acid / Q000494:pharmacology; D000469:Alkaline Phosphatase / Q000378:metabolism; D002166:Camptothecin / Q000494:pharmacology; D002467:Cell Nucleus / Q000201:enzymology; D004264:DNA Topoisomerases, Type I / Q000378:metabolism*; D004250:DNA Topoisomerases, Type II / Q000378:metabolism*; D004270:DNA, Circular / Q000737:chemistry; D004278:DNA, Superhelical / Q000737:chemistry; D009690:Nucleic Acid Conformation; D013713:Teniposide / Q000494:pharmacology; D059004:Topoisomerase I Inhibitors; D059005:Topoisomerase II Inhibitors; D003313:Zea mays / Q000196:embryology / Q000201:enzymology*
D004270:DNA, Circular; D004278:DNA, Superhelical; D059004:Topoisomerase I Inhibitors; D059005:Topoisomerase II Inhibitors; D015084:2,4-Dichlorophenoxyacetic Acid; D000040:Abscisic Acid; D013713:Teniposide; D000469:Alkaline Phosphatase; D004264:DNA Topoisomerases, Type I; D004250:DNA Topoisomerases, Type II; D002166:Camptothecin