Electrophoretic identification of poly-γ-glutamate chain-lengths of 5,10-methylenetetrahydrofolate using thymidylate synthetase complexes |
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Authors: | D.G. Priest K.K. Happel M.T. Doig |
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Affiliation: | 1. Department of Biochemistry, Medical University of South Carolina, Charleston, SC 29403, USA;2. Department of Chemistry, College of Charleston, Charleston, SC 29401, U.S.A. |
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Abstract: | A method has been developed to characterize the poly-γ-glutamates of 5,10-methyl-enetetrahydrofolate. Incorporation of 5,10-methylenetetrahydrofolates into a ternary complex with L. casei thymidylate synthetase and 5-fluoro-2-deoxy[3H]uridylate stabilizes the reduced folate against oxidation, loss of the one carbon moiety, and poly-γ-glutamate degradation. The covalent ternary complexes, containing 5,10-methylenetetrahydrofolate polyglutamates, were resolved electrophoretically. Electrophoretic mobility was shown to be a linear function of polyglutamate chain-length. The method can potentially be applied to analysis of chemically prepared folate polyglutamates, the monitoring of enzyme-mediated interconversions of polyglutamates and characterization of tissue extract polyglutamates. |
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Keywords: | folypolyglutamates thymidylate synthetase complexes FdUMP 5-fluoro-2′-deoxyuridylate EDTA ethylenediaminetetraacetic acid methylenetetrahydrofolate |
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