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Issue 1, 2014
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A luminescence-based assay of UDP-sugar producing pyrophosphorylases

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Abstract

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, 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 not only UDP-sugar producing pyrophosphorylases, but also other types of pyrophosphorylases are discussed.

Graphical abstract: A luminescence-based assay of UDP-sugar producing pyrophosphorylases

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Article information


Submitted
16 Oct 2013
Accepted
31 Oct 2013
First published
31 Oct 2013

This article is Open Access

Anal. Methods, 2014,6, 57-61
Article type
Communication

A luminescence-based assay of UDP-sugar producing pyrophosphorylases

D. Decker, S. Lindberg, J. Eriksson and L. A. Kleczkowski, Anal. Methods, 2014, 6, 57
DOI: 10.1039/C3AY41811A

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