AN IMPROVED ASSAY FOR UDPGLUCOSE PYROPHOSPHORYLASE AND OTHER ENZYMES THAT HAVE NUCLEOTIDE PRODUCTS

UDPglucose pyrophosphorylase catalyses the interconversion UDPglucose plus pyrophosphate and glucose I-phosphate plus UTP. Several assay met hods for this enzyme have been described but the only one that can be used to investigate the specificity with respect to various UDPsugars is based on coupling to UTP formation. This assay employs phosphoglyce rate kinase to catalyse the formation 1,3-bisphosphoglycerate which is then used to oxidise NADH in the presence of glyceraldehyde 3-phospha te dehydrogenase. We have found that the activity of phosphoglycerate kinase towards UTP is low which limits the usefulness of the assay to very low rates, in agreement with the published recommendation of Hans en et al.(5). Here it is shown that the dynamic range of the assay is increased by more than five fold on addition of nucleoside diphosphate kinase and ABP, which convert UTP to the preferred phosphoglycerate k inase substrate, ATP. It is also shown that the improved assay is suit able for enzymes with other nucleotide triphosphate products.