A Nonradioactive High-Performance Liquid Chromatographic Microassay for Uridine 5′-Monophosphate Synthase, Orotate Phosphoribosyltransferase, and Orotidine 5′-Monophosphate Decarboxylase

• Published in: Analytical biochemistry Vol. 299; no. 2; pp. 162 - 168
• Main Authors: Krungkrai, Jerapan, Wutipraditkul, Nuchanat, Prapunwattana, Phisit, Krungkrai, Sudaratana R., Rochanakij, Sunant
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 15.12.2001
• Subjects: Animals; Chromatography, High Pressure Liquid - methods; enzymatic assay; Erythrocytes - enzymology; high-performance liquid chromatographic assay; Humans; Kinetics; Leukocytes, Mononuclear - enzymology; mammalian cells; Mice; Multienzyme Complexes - analysis; orotate phosphoribosyltransferase; Orotate Phosphoribosyltransferase - analysis; orotidine 5′-monophosphate decarboxylase; Orotidine-5'-Phosphate Decarboxylase - analysis; uridine 5′-monophosphate synthase; orotate phosphoribosyltransferase; enzymatic assay; uridine 5′-monophosphate synthase; mammalian cells; orotidine 5′-monophosphate decarboxylase; high-performance liquid chromatographic assay
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1006/abio.2001.5431

A novel nonradioactive, microassay method has been developed to determine simultaneously the two enzymatic activities of orotate phosphoribosyltransferase (OPRTase) and orotidine 5′-monophosphate decarboxylase (ODCase), either as a bifunctional protein (uridine 5′-monophosphate synthase, UMPS) or as separate enzymes. Substrates (orotate for OPRTase or orotidine 5′-monophosphate for ODCase) and a product (UMP) of the enzymatic assay were separated by high-performance liquid chromatography (HPLC) using a reversed-phase column and an ion-pairing system; the amount of UMP was quantified by dual-wavelength uv detection at 260 and 278 nm. This HPLC assay can easily detect picomole levels of UMP in enzymatic reactions using low specific activity UMPS of mammalian cell extracts, which is difficult to do with the other nonradioactive assays that have been described. The HPLC assay is suitable for use in protein purification and for kinetic study of these enzymes.