P. Sobti et al., Radioenzymatic assay for reductive catalysis of N-5,N-10-methylenetetrahydrofolate by methylenetetrahydrofolate reductase, J BIOCH BIO, 46(1-2), 2000, pp. 11-20
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.