A direct radioassay of methylmalonyl-coenzyme A mutase using enzymatically synthesized dl-[3- 14C]methylmalonyl-CoA

Abstract

1. ( 1) dl-[3- 14C]Methylmalonyl-Coenzyme A was enzymatically synthesized from propionyl-CoA and [ 14C]NaHCO 3 (specific activity 5 μCi/μmole) employing propionyl-CoA carboxylase activity prepared from bovine liver. A net yield of 25%, based on propionyl-CoA of the purified product was achieved, having a specific activity of 4.1 μCi/μmole. 2. ( 2) Purity of the dl-[ 14C]methylmalonyl-CoA was found to be about 90% and was based on ( a) extent of decarboxylation under “back reaction conditions” of propionyl-CoA carboxylase, ( b) thin layer chromatography of the thioester, ( c) gas-liquid chromatography of the dimethylester derivative of [3- 14C]methylmalonic acid, following deacylation. 3. ( 3) The purified product was fairly stable when stored at −80°C in vacuo, undergoing decarboxylation at a rate of 4% per month. 4. ( 4) Methylmalonyl-CoA mutase activity was measured in human liver preparations employing purified dl-[3- 14C]methylmalonyl-CoA. Activity was based on the rate of formation of [ 14C]succinyl-CoA, the latter determined as the free acid isolated on thin layer chromatography. Activity was linear over a 20-minute time course in the presence of 1.25 units of enzyme and also with respect to enzyme concentration over a 1–11 unit range of enzyme activity under routine assay conditions.