Determination of mouse liver 5-methyltetrahydrofolate concentration and polyglutamate forms.
The concentration and polyglutamate status of 5-methyltetrahydrofolate in mouse liver tissue extracts has been determined by enzymatic conversion to methylenetetrahydrofolate and subsequent entrapment of this cofactor form into a ternary complex with Lactobacillus casei thymidylate synthase and tritiated 5-fluorodeoxyuridylate. 5-Methyltetrahydrofolate was oxidized to methylenetetrahydrofolate using the reverse reaction of methylenetetrahydrofolate reductase with menadione as the ultimate electron acceptor. Reference 5-methyltetrahydrofolate could be quantitatively recovered from tissue extracts by this method. The polyglutamate status of enzymatically converted and complexed tissue 5-methyltetrahydrofolate was determined electrophoretically. Unlabeled 5-fluorodeoxyuridylate was used to remove endogenous methylenetetrahydrofolate prior to enzymatic oxidation of 5-methyltetrahydrofolate and subsequent electrophoretic analysis. In this manner, the 5-methyltetrahydrofolate polyglutamate pool alone could be labeled and visualized. There were no observable differences in the polyglutamate distribution of endogenous methylenetetrahydrofolate versus 5-methyltetrahydrofolate polyglutamates in extracts of normal mouse liver tissue.
D000818:Animals; D005492:Folic Acid / Q000031:analogs & derivatives*; D008099:Liver / Q000032:analysis*; D051379:Mice; D011624:Pteroylpolyglutamic Acids / Q000032:analysis*; D013552:Swine; D013763:Tetrahydrofolates / Q000032:analysis*
D011624:Pteroylpolyglutamic Acids; D013763:Tetrahydrofolates; D005492:Folic Acid; C005984:5-methyltetrahydrofolate