Purification of Thymidylate Synthetase from Ehrlich Ascites Carcinoma Cells

Thymidylate synthetase has been purified about 4,700-fold from Ehrlich ascites carcinoma cells. The procedures used to isolate the enzyme were ammonium sulfate fractionation, DEAE-cellulose chromatography, Sephadex G-100 filtration, and chromatography on Brushite. The enzyme was demonstrated to be homogeneous by disc electrophoresis on polyacrylamide gel. By the use of gel filtration and sodium dodecyl sulfate polyacrylamide gel electrophoresis, we estimated the molecular weight of the protein to be 67,000 and 71,000 respectively. The purified enzyme did not show a requirement for Mg2+. The Km value for dl, l-methylenetetrahydrofolate was 43 µm and the Km value for deoxyuridylate was 6.3 µm. Thiols enhanced the activity of the enzyme at all stages of purification.