Radioenzymatic assay for reductive catalysis of N-5,N-10-methylenetetrahydrofolate by methylenetetrahydrofolate reductase

Methylenetetrahydrofolate reductase catalyzes the reduction of N-5,N-10-met hylenetetrahydrofolate to N-5-methyltetrahydrofolate. Because this substrat e is unstable and dissociates spontaneously into formaldehyde and tetrahydr ofolate, the customary method to assay the catalytic activity of this enzym e has been to measure the oxidation of [C-14]N-5-methyltetrahydrofolate to N-5,N-10-methylenetetrahydrofolate and quantify the [C-14]formaldehyde that dissociates from this product. This report describes a very sensitive radi oenzymatic assay that measures directly the reductive catalysis of N-5,N-10 -methylenetetrahydrofolate. The radio-labeled substrate, [C-14](NN10)-N-5-m ethylenetetrahydrofolate, is prepared by condensation of [C-14]formaldehyde with tetrahydrofolate and the stability of this substrate is maintained fo r several months by storage at - 80 degreesC 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, ge nerates [C-14]N-5 methyltetrahydrofolate, which is stable and partitions in to the aqueous phase after the assay is terminated with dimedone and toluen e. A K-m value of 8.2 muM was obtained under conditions of increasing subst rate concentration to ensure saturation kinetics. This method is simple, ve ry sensitive and measures directly the reduction of N-5,N-10-methylenetetra hydrofolate to N-5-methyltetrahydrofolate, which is the physiologic catalyt ic pathway for methylenetetrahydrofolate reductase. (C) 2000 Elsevier Scien ce B.V. All rights reserved.