Abstract
Publisher Summary This chapter discusses the enzymatic spectrophotometric assay to determine uridine-5-triphosphate (UTP), uridine-5-diphosphate (UDP), and uridine-5-monophosphate (U-5-MP). The irreversible oxidation of UDP-glucose to UDP glucuronate, in which 2 moles of NAD are reduced per mole of UDP glucose by UDP glucose dehydrogenase (UDPG-DH), is used as the indicator reaction. In the presence of glucose-1-phosphate, UTP is converted into UDP glucose and pyrophosphate with the help of enzyme, UDP-glucose pyrophosphorylase (UDPGP). The combined reactions are highly specific for uridinephosphate. UDP can be converted into UTP with the help of nucleoside diphosphate kinase (NDPK). U-5-MP is phosphorylated to UDP with the help of ATP by nucleoside monophosphate kinase (NMPK). The method has application in biochemistry. The NAD reduction measured sequentially at 340 (334, 365) nm is proportional to the quantity of UDPglucose, UTP, UDP, and/or U-5-MP. The pH optimum for the determination of U-5-MP, UDP, and UTP with UDPG dehydrogenase as the indicator enzyme is pH 8.7. In contrast to UDPG dehydrogenase, which is active even in the presence of ethylenediaminetetraacetic acid (EDTA), UDPG pyrophosphorylase requires divalent cations; the highest activity is exhibited by magnesium.
Published Version
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