A luminescence-based assay of UDP-sugar producing pyrophosphorylases
2014 (English)In: Analytical Methods, ISSN 1759-9660, Vol. 6, no 1, 57-61 p.Article in journal (Refereed) Published
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 neither affected by Pi nor by DTT. For UGPase, K-m values with UTP and Glc-1-P were 0.14 and 0.26 mM, respectively, whereas for USPase the respective K-m 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 not only UDP-sugar producing pyrophosphorylases, but also other types of pyrophosphorylases are discussed.
Place, publisher, year, edition, pages
RSC Publishing, 2014. Vol. 6, no 1, 57-61 p.
Chemical Sciences Botany
IdentifiersURN: urn:nbn:se:umu:diva-85622DOI: 10.1039/c3ay41811aISI: 000329071500004OAI: oai:DiVA.org:umu-85622DiVA: diva2:695139
FunderSwedish Research Council