Isolation of the covalent binary complex of 5-fluorodeoxyuridylate and thymidylate synthetase by trichloroacetic acid precipitation |
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Authors: | M A Moore F Ahmed R B Dunlap |
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Institution: | Department of Chemistry University of South Carolina Columbia, South Carolina 29208 USA |
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Abstract: | 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. |
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Keywords: | FdUMP 5-fluorodeoxyuridylate (±)-5 10-methylenetetrahydrofolate NEM N-ethylmaleimide TCA trichloroacetic acid DPM disintegrations per minute |
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