Purification and characterization of type II DNA topoisomerase from mouse FM3A cells: phosphorylation of topoisomerase II and modification of its activity.
Type II topoisomerase has been purified from mouse FM3A cells by using P4 phage knotted DNA as a substrate. Analysis of the purified enzyme by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed two bands of apparent molecular masses of 167 and 151 kDa. Partial digestion of the two bands with Staphylococcus aureus V8 protease indicated that the two polypeptides were structurally related. The enzyme required ATP and Mg2+ for activity. dATP could substitute for ATP, and ITP was slightly effective at 5-10 mM. The activity was sensitive to 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA), coumermycin, and ethidium bromide. A protein kinase activity was detected in the partially purified topoisomerase II fraction, and this protein kinase was further purified. The protein kinase phosphorylated the purified topoisomerase II, and the phosphorylation of topoisomerase II by the kinase increased the activity by 8.6-fold over that of the unmodified enzyme. The treatment of the purified topoisomerase II with alkaline phosphatase abolished the enzyme activity almost completely, and the treatment of the dephosphorylated topoisomerase II with the protein kinase restored the enzyme activity. The protein kinase activity was not stimulated by Ca2+ or cyclic nucleotides, and the aminoacyl residue phosphorylated by the kinase was serine. Enzymatic properties of the kinase were very similar to those of the kinase reported to be tightly associated with the Drosophila topoisomerase II [Sander, M., Nolan, J. M., & Hsieh, T.-S. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 6938-6942]. The immunoprecipitation of nuclear extracts prepared from 32P-labeled cells with anti-mouse topoisomerase II antiserum indicated that DNA topoisomerase II existed in mouse cells as a phosphoprotein.
Saijo
M
M
Department of Physiological Chemistry, Faculty of Pharmaceutical Sciences, University of Tokyo, Japan.
Enomoto
T
T
Hanaoka
F
F
Ui
M
M
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000469:Alkaline Phosphatase / Q000494:pharmacology; D000818:Animals; D004250:DNA Topoisomerases, Type II / Q000302:isolation & purification / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D007163:Immunosorbent Techniques; D008274:Magnesium / Q000494:pharmacology; D008325:Mammary Neoplasms, Experimental; D051379:Mice; D008809:Mice, Inbred C3H; D008970:Molecular Weight; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism; D011494:Protein Kinases / Q000378:metabolism; D012697:Serine Endopeptidases / Q000378:metabolism; D059005:Topoisomerase II Inhibitors; D014407:Tumor Cells, Cultured
D059005:Topoisomerase II Inhibitors; D010768:Phosphoserine; D000255:Adenosine Triphosphate; D011494:Protein Kinases; D000469:Alkaline Phosphatase; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase; D004250:DNA Topoisomerases, Type II; D008274:Magnesium