Determination of inorganic phosphate by coupling thymidine phosphorylase and reversed-phase high-performance liquid chromatography: application to tonoplast pyrophosphatase activity.
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.
Hill
M
M
ERS 571 du CNRS, Bioénergétique Membranaire, Université de Paris Sud, Centre d'Orsay, France.
Arrio
B
B
D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D010710:Phosphates / Q000032:analysis* / Q000378:metabolism; D010940:Plant Proteins / Q000378:metabolism; D010944:Plants / Q000201:enzymology; D013936:Thymidine / Q000378:metabolism; D013939:Thymidine Phosphorylase / Q000378:metabolism; D013941:Thymine / Q000378:metabolism
D010710:Phosphates; D010940:Plant Proteins; D013939:Thymidine Phosphorylase; D013941:Thymine; D013936:Thymidine