Possibilities of enzyme synthesis of radioactive nucleotides. V

A new model of the enzymic synthesis of radioactive orotidine-5'-monophosphate of high molar radioactivity labelled with 14C or 3H was developed. The synthesis is based on the specific properties of cell-free extracts of Brevibacterium ammoniagenes CCEB 364 which catalyze the conversion of orotic acid to orotidine-5'-monophosphate in the presence of 5-phosphoribosyl-1-pyrophosphate as the donor of phosphoribosyl groups and of 6-azauridine-5'-monophosphate as the inhibitor of orotidine-5'-phosphate decarboxylase. As the cell-free extract contains no 5'-nucleotidase, the generated orotidine-5'-phosphate is not dephosphorylated and the high yields are thereby attained. The reaction conditions were studied in order to achieve maximum production of radioactive orotidine-5'-monophosphate and the feasibility of separating this product from the reaction mixture by preparative paper chromatography and its chemical and radiochemical stability were investigated. [2-14C]orotidine-5'-monophosphate and [7-14C]orotidine-5'-monophosphate with a molar radioactivity of 1400 to 1600 MBq.mmol-1 and radiochemical purity of 96 to 98%, and [5-3H]orotidine-5'-monophosphate with a molar radioactivity of 580 GBq.mmol-1 and radiochemical purity of 98% were prepared on a preparative scale. (author)