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Anal Biochem. 1997 Dec 15;254(2):249-53.

Determination of inorganic phosphate by coupling thymidine phosphorylase and reversed-phase high-performance liquid chromatography: application to tonoplast pyrophosphatase activity.

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ERS 571 du CNRS, Bioénergétique Membranaire, Université de Paris Sud, Centre d'Orsay, France.


We developed an HPLC method for the measurement of inorganic phosphate using thymidine phosphorylase (EC 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.

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