Abstract
Thymidylate synthetase from HeLa cells was purified to electrophoretical homogenity as a result of affinity chromatography on a 10-formyl-5,8-dideazafolate-ethyl-Sepharose column. Electrophoretical analysis of the formation of the enzyme-5-fluorodeoxyuridylate-5,10-methylenetetrahydrofolate complex shows the presence of two binding sites for 5-fluorodeoxyuridylate on the enzyme molecule. The molecular weight of the enzyme subunit was 36,000. The apparent Michaelis constants for dUMP and ( ± )-
l-5,10-methylenetetrahydrofolate were 2.0 and 31 μM, respectively. The enzyme exhibited a temperature-dependent conformational change with a transitional temperature of 35°. Activation energies above and below this temperature were 8.1 and 20.3 kcal/mole, respectively.