Rapid purification and characterization of DNA topoisomerase I from cultured mouse mammary carcinoma FM3A cells.

Abstract

We have previously shown that a DNA topoisomerase I from mouse mammary carcinoma cells is inhibited by heparin.Taking advantage of this enzyme-heparin interaction, we developed a rapid and efficient method of purification of this enzyme to near homogeneity by extraction of chromatin with 0.15 M phosphate buffer followed by two-step column chromatography on heparin-Sepharose and phenyl-Sepharose.Electrophoresis on sodium dodecyl sulfate-polyacrylamide gels revealed that the final preparation is composed of two polypeptides with apparent M , -98,000 (p98) and 102,000 (p102), p98 comprising 70% and p102 30%.Extraction and renaturation of the polypeptides from the gel shows that both p98 and p102 seem to possess topoisomerase activity.Partial proteolytic digestion of p98 and p102 with Staphylococcus aureus V8 and chymotrypsin yielded a series of identical peptides, indicating that the two polypeptides are structurally related.The enzyme sedimented through sucrose density gradient with s20,w of 4.0 s, and thus is monomeric in solution.An enzyme activity from mammalian cells that removes positive and negative superhelical turns from DNA was first described by Champoux and Dulbecco (1972).Similar enzymes were subsequently found in various eukaryotic organisms largely in association with chromatin (Champoux, 1978;Wang and Liu, 1979).The physiological functions of type I