Thymidylate synthetase purified to homogeneity from human leukemic cells.

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Abstract
Thymidylate synthetase (5,10-methylenetetrahydrofolate:dUMP C-methyltransferase, EC 2.1.1.45) from a human leukemic cell line was purified to homogeneity with 1-step affinity column chromatography. The purified enzyme has a specific activity of 3.8 .mu.mol/min per mg of protein, which corresponds to a turnover number of 250. These are the highest values reported for a thymidylate synthetase from neoplastic tissue. A ratio of 1.7 mol of 5-fluoro-2''-deoxyuridylate binds per mol of enzyme in the presence of 5,10-methylenetetrahydrofolate. The ternary complex so formed migrates intact on denaturing gels and can be precipitated with trichloroacetic acid; however, urea dissociates the ternary complex. The human thymidylate synthetase is composed of 2 subunits of 33,000 daltons each. It contains more residues of cysteine, glycine and arginine and fewer of histidine than the well-studied thymidylate synthetase from Lactobacillus casei.