title: Purification of mammalian tumor (L1210) thymidylate synthetase by affinity chromatography on stable biospecific adsorbent. Stabilization of the enzyme with neutral detergents.
abstract: Thymidylate synthetase from mouse leukemic L1210 cells was purified to electrophoretic homogeneity with 70% yield as a result of an affinity chromatography procedure based on reversible deoxyuridylate-dependent binding of the enzyme to a stable biospecific adsorbent, 10-formyl-5,8-dideazafolate, immobilized on aminoethyl-Sepharose. The presence of neutral detergents, Triton X-100, or Nonidet P40 stabilized thymidylate synthetase during purification. Analytical electrophoresis of the enzyme treated with an excess of 5-fluorodeoxyuridylate and 5,10-methylenetetrahydrofolate showed the presence of two forms of thymidylate synthetase--5-fluorodeoxyuridylate.5,10-methylenetetrahydrofolate complex, indicating that there are two binding sites for 5-fluorodeoxyuridylate present on the enzyme molecule. Molecular weight of native thymidylate synthetase was found to be 75,000, whereas that for the monomer was 38,500.
mesh_terms: D000818:Animals; D002846:Chromatography, Affinity; D003902:Detergents; D004355:Drug Stability; D004591:Electrophoresis, Polyacrylamide Gel; D007939:Leukemia L1210 / Q000201:enzymology*; D008780:Methyltransferases / Q000302:isolation & purification*; D051379:Mice; D008970:Molecular Weight; D013940:Thymidylate Synthase / Q000302:isolation & purification*
chemical_list: D003902:Detergents; D008780:Methyltransferases; D013940:Thymidylate Synthase