A Facile Fluorometric Assay of Orotate Phosphoribosyltransferase Activity Using a Selective Fluorogenic Reaction for Orotic Acid.

ABSTRACT: Orotate phosphoribosyltransferase (OPRT) exists as a bifunctional enzyme, uridine 5'-monophosphate synthase, in mammalian cells and plays an important role in pyrimidine biosynthesis. Measuring OPRT activity has been considered important for understanding biological events and development of molecular-targeting drugs. In this study, we demonstrate a novel fluorescence method for measuring OPRT activity in living cells. The technique utilizes 4-trifluoromethylbenzamidoxime (4-TFMBAO) as a fluorogenic reagent, which produces selective fluorescence for orotic acid. To perform the OPRT reaction, orotic acid was added to HeLa cell lysate, and a portion of the enzyme reaction mixture was heated at 80 °C for 4 min in the presence of 4-TFMBAO under basic conditions. The resulting fluorescence was

SUBMITTER: Shibata T 

PROVIDER: S-EPMC10007123 | biostudies-literature | 2023 Feb

REPOSITORIES: biostudies-literature