Purification of topoisomerase II from amsacrine-resistant P388 leukemia cells. Evidence for two forms of the enzyme

Topoisomerase II was purified from an amsacrine-resistant mutant of P388 leukemia. A procedure has been developed which allows the rapid purification of nearly homogeneous enzyme in quantities sufficient for enzyme studies or production of specific antisera. The purified topoisomerase II migrated on...

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Bibliographic Details
Published in:The Journal of biological chemistry 1987-12, Vol.262 (34), p.16739-16747
Main Authors: Drake, FH, Zimmerman, JP, McCabe, FL, Bartus, HF, Per, SR, Sullivan, DM, Ross, WE, Mattern, MR, Johnson, RK, Crooke, ST
Format: Article
Language:English
Subjects:
Amsacrine - therapeutic use
Analytical, structural and metabolic biochemistry
Animals
Biological and medical sciences
DNA - metabolism
DNA topoisomerase (ATP-hydrolysing)
DNA Topoisomerases, Type II - isolation & purification
Drug Resistance
Enzymes and enzyme inhibitors
Fundamental and applied biological sciences. Psychology
Immunosorbent Techniques
Isoenzymes - isolation & purification
Isomerases
Leukemia P388 - drug therapy
Leukemia, Experimental - drug therapy
Nucleic Acid Conformation
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Summary:Topoisomerase II was purified from an amsacrine-resistant mutant of P388 leukemia. A procedure has been developed which allows the rapid purification of nearly homogeneous enzyme in quantities sufficient for enzyme studies or production of specific antisera. The purified topoisomerase II migrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as two bands with apparent molecular masses of 180 (p180) and 170 kDa (p170); both proteins unknotted P4 DNA in an ATP-dependent manner and displayed amsacrine-stimulated covalent attachment to DNA. Staphylococcus V8 protease cleavage patterns of p170 and p180 showed distinct differences. Specific polyclonal antibodies to either p170 or p180 recognized very selectively the form of the enzyme used to generate the antibodies. Immunoblotting with these specific antibodies showed that both p180 and p170 were present in cells lysed immediately in boiling sodium dodecyl sulfate. Comparison of the purified topoisomerase II from amsacrine-resistant P388 with that from amsacrine-sensitive P388 demonstrated that each cell type contained both p180 and p170; however, the relative amounts of the two proteins were consistently different in the two cell types. The data strongly suggest that p170 is not a proteolytic fragment of p180. Thus, P388 cells appear to contain two distinct forms of topoisomerase II.
ISSN:0021-9258
1083-351X
DOI:10.1016/S0021-9258(18)49317-9