A luminescence-based assay of UDP-sugar producing pyrophosphorylases
Anal. Methods, 2014, 6(1):57-61 Decker D, Lindberg S, Eriksson J, Kleczkowski LA
A coupled luminescence assay was applied to monitor pyrophosphate (PPi) production by either purified barley UDP-glucose pyrophosphorylase (UGPase) or purified Leishmania UDP-sugar pyrophosphorylase (USPase). In the assay, the PPi produced by the pyrophosphorylases was converted to ATP by ATP-sulfurylase, and the ATP produced was linked to luminescent light formation through the action of firefly luciferase. The assay allowed for a quantitative measurement of UGPase and USPase activities, down to a pmol per min level. The activities were linear with time and proportional to the amount of the enzyme added, and were not affected by Pi nor DTT. For UGPase, Km values with UTP and Glc-1-P were 0.14 and 0.26 mM, respectively, whereas for USPase the respective Km values with UTP, Glc-1-P and Gal-1-P were 0.4, 2.9 and 3.9 mM. Possible applications of the luminescence-based assay for UDP-sugar producing pyrophosphorylases, but also other types of pyrophosphorylases, are discussed.
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