Isolation of the covalent binary complex of 5-fluorodeoxyuridylate and thymidylate synthetase by trichloroacetic acid precipitation.
Strong chemical evidence for the existence of a covalent binary complex between 5-fluorodeoxyuridylate and thymidylate synthetase was provided by the isolation of the complex by trichloroacetic acid precipitation. This result together with that of a control experiment with N-ethymaleimide inactivated thymidylate synthetase demonstrated that only nucleotide covalently bound to the protein survived repeated washings of the precipitate. Under the conditions used, a maximum binding stoichiometry of about 0.9 was obtained for the covalent binary complex, Kd = 1.1 X 10(-5) M. Also, a binding ratio of 1.7 was obtained for the methylenetetrahydrofolate-5-fluorodeoxyuridylate-thymidylate synthetase ternary complex.
D003856:Deoxyuracil Nucleotides / Q000302:isolation & purification*; D005468:Fluorodeoxyuridylate / Q000302:isolation & purification* / Q000378:metabolism; D007700:Kinetics; D007780:Lacticaseibacillus casei / Q000201:enzymology; D008780:Methyltransferases / Q000302:isolation & purification*; D011485:Protein Binding; D013940:Thymidylate Synthase / Q000302:isolation & purification* / Q000378:metabolism; D014238:Trichloroacetic Acid; D014316:Tritium
D003856:Deoxyuracil Nucleotides; D014316:Tritium; D005468:Fluorodeoxyuridylate; D014238:Trichloroacetic Acid; D008780:Methyltransferases; D013940:Thymidylate Synthase