Rg. Duggleby et al., AN IMPROVED ASSAY FOR UDPGLUCOSE PYROPHOSPHORYLASE AND OTHER ENZYMES THAT HAVE NUCLEOTIDE PRODUCTS, Experientia, 52(6), 1996, pp. 568-572
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.