Isolation of a homogeneous preparation of human thymidylate synthetase from HeLa cells.
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 microM, respectively. The enzyme exhibited a temperature-dependent conformational change with a transitional temperature of 35 degrees . Activation energies above and below this temperature were 8.1 and 20.3 kcal/mole, respectively.
Rode
W
W
Department of Pharmacology, Yale University School of Medicine, New Haven, CT 06510, USA.
Dolnick
B J
BJ
Bertino
J R
JR
D005468:Fluorodeoxyuridylate / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D011485:Protein Binding; D013763:Tetrahydrofolates / Q000378:metabolism; D013940:Thymidylate Synthase / Q000302:isolation & purification*
D013763:Tetrahydrofolates; C013123:5,10-methylenetetrahydrofolic acid; D005468:Fluorodeoxyuridylate; D013940:Thymidylate Synthase