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 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.
Drake
F H
FH
Department of Molecular Pharmacology, Smith Kline and French Laboratories, Philadelphia, Pennsylvania 19101.
Zimmerman
J P
JP
McCabe
F L
FL
Bartus
H F
HF
Per
S R
SR
Sullivan
D M
DM
Ross
W E
WE
Mattern
M R
MR
Johnson
R K
RK
Crooke
S T
ST
D000677:Amsacrine / Q000627:therapeutic use*; D000818:Animals; D004247:DNA / Q000378:metabolism; D004250:DNA Topoisomerases, Type II / Q000302:isolation & purification*; D004351:Drug Resistance; D007163:Immunosorbent Techniques; D007527:Isoenzymes / Q000302:isolation & purification*; D007941:Leukemia P388 / Q000188:drug therapy*; D007942:Leukemia, Experimental / Q000188:drug therapy*; D009690:Nucleic Acid Conformation
D007527:Isoenzymes; D000677:Amsacrine; D004247:DNA; D004250:DNA Topoisomerases, Type II