A coupled radiometric assay for dihydrofolate reductase based on the oxidative decarboxylation of D-6-phospho-[1-14C]gluconate.
The synthesis of D-6-phospho-[1-14C]gluconate and the utilization of this compound in a novel assay procedure for dihydrofolate reductase is described. This new assay method couples reductase-dependent NADP+ production to the enzymatic and NADP+-dependent decarboxylation of D-6-phospho-[1-14C]gluconate. By several criteria it is demonstrated that [14C]CO2 release is directly proportional to dihydrofolate reductase activity. This coupled radiometric assay for dihydrofolate reductase is more sensitive than the commonly used spectrophotometric assay and offers a number of advantages over earlier radiometric methods.
D000818:Animals; D002417:Cattle; D003653:Decarboxylation; D005942:Gluconates / Q000138:chemical synthesis; D011874:Radiometry / Q000379:methods; D013056:Spectrophotometry, Ultraviolet; D013762:Tetrahydrofolate Dehydrogenase / Q000032:analysis*
D005942:Gluconates; D013762:Tetrahydrofolate Dehydrogenase; C008884:6-phosphogluconic acid