Evaluation of folylpolyglutamates by electrophoretic separation of fluorodeoxyuridylate-thymidylate synthase-methylenetetrahydrofolate complexes.
The chain length of specific reduced folylpolyglutamates has been estimated by incorporation of the 5,10-methylenetetrahydrofolate form of the cofactor into a stable ternary complex with L. casei thymidylate synthase and tritiated fluorodeoxyuridylate followed by electrophoretic separation based on charge differences in complexed polyglutamates. The method is also applicable to tetrahydrofolate polyglutamates after conversion to the active cofactor form by introduction of formaldehyde. The method can be used to analyze less than one pmole of folylpolyglutamate and can be applied to evaluation of tissue polyglutamates or to monitor relevant enzyme catalyzed reactions.
D000818:Animals; D003856:Deoxyuracil Nucleotides / Q000302:isolation & purification*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D005468:Fluorodeoxyuridylate / Q000302:isolation & purification* / Q000378:metabolism; D005492:Folic Acid / Q000031:analogs & derivatives*; D007700:Kinetics; D007780:Lacticaseibacillus casei / Q000201:enzymology*; D008099:Liver / Q000201:enzymology*; D008780:Methyltransferases / Q000302:isolation & purification*; D051379:Mice; D011485:Protein Binding; D011624:Pteroylpolyglutamic Acids / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D013045:Species Specificity; D013763:Tetrahydrofolates / Q000302:isolation & purification* / Q000378:metabolism; D013940:Thymidylate Synthase / Q000302:isolation & purification* / Q000378:metabolism
D003856:Deoxyuracil Nucleotides; D011624:Pteroylpolyglutamic Acids; D013763:Tetrahydrofolates; C013123:5,10-methylenetetrahydrofolic acid; D005468:Fluorodeoxyuridylate; D005492:Folic Acid; D008780:Methyltransferases; D013940:Thymidylate Synthase