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

Anal Biochem. 1997 Dec 15;254(2):249-53. doi: 10.1006/abio.1997.2437.

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.

MeSH terms

  • Chromatography, High Pressure Liquid / methods*
  • Phosphates / analysis*
  • Phosphates / metabolism
  • Plant Proteins / metabolism
  • Plants / enzymology
  • Thymidine / metabolism
  • Thymidine Phosphorylase / metabolism
  • Thymine / metabolism

Substances

  • Phosphates
  • Plant Proteins
  • Thymidine Phosphorylase
  • Thymine
  • Thymidine