A high molecular weight topoisomerase I from Xenopus laevis ovaries.
DNA topoisomerase I has been purified from homogenates of mature Xenopus laevis ovaries. The initial stages in purification of the native enzyme employed a rapid series of three chromatographic steps, followed by gel filtration performed in the presence of sodium dodecyl sulfate. Polypeptides that might represent topoisomerase I were identified by specific labeling of the topoisomerase species with radioactive DNA. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of topoisomerase I radiolabeled with DNA identified three polypeptides with mobilities consistent with sizes of 165, 125, and 88 kDa. All three polypeptides were found to possess topoisomerase activity following elution from the gel and renaturation. Partial proteolytic digestion of the radiolabeled 165-, 125-, and 88-kDa polypeptides with Staphylococcus aureus V8 endoproteinase resulted in identical autoradiographic patterns. This suggests that the 125-kDa and 88-kDa polypeptides may be degradation products of the 165-kDa species. The 165-kDa topoisomerase I exhibited the same sensitivity to camptothecin as the total, native topoisomerase I fraction.
Richard
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Department of Pharmacological Sciences, State University of New York, Stony Brook 11794-8651.
Bogenhagen
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D000818:Animals; D002166:Camptothecin / Q000494:pharmacology; D002850:Chromatography, Gel; D004264:DNA Topoisomerases, Type I / Q000302:isolation & purification* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D008970:Molecular Weight; D010053:Ovary / Q000201:enzymology*; D010446:Peptide Fragments / Q000378:metabolism; D011489:Protein Denaturation; D012697:Serine Endopeptidases / Q000378:metabolism; D012967:Sodium Dodecyl Sulfate; D059004:Topoisomerase I Inhibitors; D014982:Xenopus laevis
D010446:Peptide Fragments; D059004:Topoisomerase I Inhibitors; D012967:Sodium Dodecyl Sulfate; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase; D004264:DNA Topoisomerases, Type I; D002166:Camptothecin