Quantification of uridine 5 ′-diphosphate (UDP)–glucose by high-performance liquid chromatography and its application to a nonradioactive assay for nucleoside diphosphate kinase using UDP–glucose pyrophosphorylase as a coupling enzyme

We describe a method for the detection and quantification of nucleoside diphosphate kinase (NDPK). NDPK catalyzes the transfer of the γ-phosphate of cytidine 5 ′-triphosphate on uridine 5 ′-diphosphate (UDP) to produce uridine 5 ′-triphosphate (UTP). The method uses a nonradioactive coupled enzyme assay in which UTP produced by NDPK is utilized by UDP–glucose pyrophosphorylase. This latter enzyme synthesizes UDP–glucose and inorganic phosphate in the presence of glucose 1-phosphate. UDP–glucose is detected at 260 nm after separation of the reaction mixture by high-performance liquid chromatography (HPLC) on a strong anion-exchange column. The assay is reliable, specific, and linear with respect to time and enzyme amount. Using 15 min incubation time, the method allows detection of NDPK activity below 10 pmol/min. It can be used to analyze kinetic behavior and to quantify NDPK from a wide variety of animal, microbial, and plant sources. It also provides an alternative to radiometric assays and an improvement on pyruvate kinase-linked spectrophotometric assays, which can be hampered by pigments present in crude extracts. Furthermore, we show that the HPLC method developed here can be directly used to assay enzymes for which UDP–glucose is a product.