Biochemical and pharmacological properties of p170 and p180 forms of topoisomerase II.
The p170 and p180 forms of topoisomerase II have been compared. The concentration dependence of ATP for catalytic activity of the two forms of the enzyme was identical, and each was equally sensitive to novobiocin. Orthovanadate was found to be a potent inhibitor of catalytic activity of both p170 and p180, with an IC50 value of about 2 microM for each. Under standard reaction conditions, relaxation of supercoiled pBR322 by p180 was highly processive, while p170 performed the same reaction in a distributive manner. The optimal concentration of KCl for catalytic activity of p180 was 20-30 mM higher than that for p170. Comparison of their thermal stability showed that p180 was inactivated at twice the rate of p170. Teniposide and merbarone selectively inhibited catalytic activity of p170, requiring concentrations 3-fold and 8-fold lower, respectively, than those required for equivalent inhibition of p180. Similar selectivity for p170 was seen for teniposide-stimulated DNA cleavage or its inhibition by merbarone. Analysis of sites of DNA cleavage indicated a subset of sites that were either preferred or unique for each of the enzymes. A synthetic oligonucleotide representative of p170 sites selectively inhibited the p170 enzyme. Immunoblotting of p170 and p180 from U937 cells at different stages of proliferation showed that p170 levels declined as the cells reached the plateau phase of growth, while p180 levels were low during rapid proliferation and increased as the growth rate slowed. The data indicate that the p170 and p180 forms of topoisomerase II can be distinguished biochemically, pharmacologically, and by differential cellular regulation.
Drake
F H
FH
Department of Molecular Pharmacology, Smith Kline and French Laboratories, King of Prussia, Pennsylvania 19406.
Hofmann
G A
GA
Bartus
H F
HF
Mattern
M R
MR
Crooke
S T
ST
Mirabelli
C K
CK
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology; D002384:Catalysis; D002478:Cells, Cultured; D004247:DNA / Q000378:metabolism*; D004250:DNA Topoisomerases, Type II / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D015151:Immunoblotting; D008970:Molecular Weight; D009690:Nucleic Acid Conformation; D010957:Plasmids; D059005:Topoisomerase II Inhibitors
D000970:Antineoplastic Agents; D059005:Topoisomerase II Inhibitors; D000255:Adenosine Triphosphate; D004247:DNA; D004250:DNA Topoisomerases, Type II