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A Purification and In Vitro Activity Assay for a (p)ppGpp Synthetase from Clostridium difficile
Published on: November 3, 2018
Determination of inorganic phosphate by coupling thymidine phosphorylase and reversed-phase high-performance liquid
1ERS 571 du CNRS, Bioénergétique Membranaire, Université de Paris Sud, Centre d'Orsay, France.
Abstract:
We developed an HPLC method for the measurement of inorganic phosphate using thymidine phosphorylase (EC 2.4.2.4). This enzyme catalyzes the phosphorolysis of thymidine to 2-deoxyribose 1-phosphate and thymine. Thymine release was measured at 265 nm after separation by reverse-phase HPLC. The assay was sensitive enough to detect as little as 10 pmol of Pi. The response to the phosphate concentration was linear from 1 to 100 microM. The value of this method was demonstrated in an analysis of the kinetics of Pi release from PPi in the presence of Catharanthus roseus tonoplast pyrophosphatase.

