A luminescence-based assay of UDP-sugar producing pyrophosphorylases

Decker, Daniel (författare): Umeå universitet,Institutionen för fysiologisk botanik,Umeå Plant Science Centre (UPSC)

Kleczkowski, Leszek A. (författare): Umeå universitet,Institutionen för fysiologisk botanik,Umeå Plant Science Centre (UPSC)

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Abstract Ämnesord

Stäng

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.

Av författaren/redakt...: Decker, Daniel; Lindberg, Stina; Eriksson, Jonas; Kleczkowski, Les ...

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