An improved assay for UDPglucose pyrophosphorylase and other enzymes that have nucleotide products.
UDPglucose pyrophosphorylase catalyses the interconversion UDPglucose plus pyrophosphate and glucose 1-phosphate plus UTP. Several assay methods 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 phosphoglycerate kinase to catalyse the formation 1,3- bisphosphoglycerate which is then used to oxidise NADH in the presence of glyceraldehyde 3-phosphate 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 Hansen et al. 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 ADP, which convert UTP to the preferred phosphoglycerate kinase substrate, ATP. It is also shown that the improved assay is suitable for enzymes with other nucleotide triphosphate products.
Duggleby
R G
RG
Department of Biochemistry, University of Queensland, Brisbane, Australia. duggleby@biosci.uq.edu.au
Peng
H L
HL
Chang
H Y
HY
D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D005958:Glucosephosphates / Q000378:metabolism; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D009243:NAD / Q000378:metabolism; D009711:Nucleotides / Q000378:metabolism*; D010710:Phosphates / Q000378:metabolism; D010735:Phosphoglycerate Kinase / Q000378:metabolism; D005957:UTP-Glucose-1-Phosphate Uridylyltransferase / Q000032:analysis* / Q000378:metabolism*; D014532:Uridine Diphosphate Glucose / Q000378:metabolism; D014544:Uridine Triphosphate / Q000378:metabolism
D005958:Glucosephosphates; D009711:Nucleotides; D010710:Phosphates; D009243:NAD; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; C031590:glucose-1-phosphate; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D010735:Phosphoglycerate Kinase; D005957:UTP-Glucose-1-Phosphate Uridylyltransferase; D014544:Uridine Triphosphate; D014532:Uridine Diphosphate Glucose