Radioenzymatic assay for reductive catalysis of N(5)N(10)-methylenetetrahydrofolate by methylenetetrahydrofolate reductase.
Methylenetetrahydrofolate reductase catalyzes the reduction of N(5), N(10)-methylenetetrahydrofolate to N(5)-methyltetrahydrofolate. Because this substrate is unstable and dissociates spontaneously into formaldehyde and tetrahydrofolate, the customary method to assay the catalytic activity of this enzyme has been to measure the oxidation of [14C]N(5)-methyltetrahydrofolate to N(5), N(10)-methylenetetrahydrofolate and quantify the [14C]formaldehyde that dissociates from this product. This report describes a very sensitive radioenzymatic assay that measures directly the reductive catalysis of N(5),N(10)-methylenetetrahydrofolate. The radio-labeled substrate, [14C]N(5),N(10)-methylenetetrahydrofolate, is prepared by condensation of [C(14)]formaldehyde with tetrahydrofolate and the stability of this substrate is maintained for several months by storage at -80 degrees C in a pH 9.5 buffer. Partially purified methylenetetrahydrofolate reductase from rat liver, incubated with the radio-labeled substrate and the cofactors, NADPH and FAD at pH 7. 5, generates [14C]N(5)-methyltetrahydrofolate, which is stable and partitions into the aqueous phase after the assay is terminated with dimedone and toluene. A K(m) value of 8.2 microM was obtained under conditions of increasing substrate concentration to ensure saturation kinetics. This method is simple, very sensitive and measures directly the reduction of N(5), N(10)-methylenetetrahydrofolate to N(5)-methyltetrahydrofolate, which is the physiologic catalytic pathway for methylenetetrahydrofolate reductase.
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Division of Hematology/Oncology, Departments of Medicine and Biochemistry, SUNY-Downstate Medical Center, Brooklyn, NY, USA.
Rothenberg
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Quadros
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D000818:Animals; D002250:Carbon Radioisotopes; D002384:Catalysis; D002855:Chromatography, Thin Layer; D004305:Dose-Response Relationship, Drug; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D042965:Methylenetetrahydrofolate Reductase (NADPH2); D009249:NADP / Q000378:metabolism; D010084:Oxidation-Reduction; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000032:analysis* / Q000302:isolation & purification / Q000378:metabolism; D051381:Rats; D012435:S-Adenosylhomocysteine / Q000378:metabolism; D012436:S-Adenosylmethionine / Q000378:metabolism; D012680:Sensitivity and Specificity; D013763:Tetrahydrofolates / Q000138:chemical synthesis / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D002250:Carbon Radioisotopes; D013763:Tetrahydrofolates; C013123:5,10-methylenetetrahydrofolic acid; D005182:Flavin-Adenine Dinucleotide; D009249:NADP; D012436:S-Adenosylmethionine; D012435:S-Adenosylhomocysteine; D000587:Oxidoreductases Acting on CH-NH Group Donors; D042965:Methylenetetrahydrofolate Reductase (NADPH2); C005984:5-methyltetrahydrofolate