A screening method for phosphohistidine phosphatase 1 activity

  • Ulla Beckman-Sundh Department of Medical Biochemistry and Microbiology, Uppsala University, Uppsala, Sweden; and Toxicology Division, National Food Administration, Uppsala, Sweden
  • Bo Ek Department of Physical and Analytical Chemistry, Uppsala University, Uppsala, Sweden; and Science for Life Laboratory, Department of Medical Biochemistry and Microbiology, Uppsala University, Uppsala, Sweden
  • Örjan Zetterqvist Department of Medical Biochemistry and Microbiology, Uppsala University, Uppsala, Sweden
  • Pia Ek of Medical Biochemistry and Microbiology, Uppsala University, Uppsala, Sweden
Keywords: Activity, assay, phosphohistidine phosphatase, PHPT1, protein histidine phosphatase

Abstract

Introduction. Research in the field of protein-bound phosphohistidine phosphorylation has been hampered by the difficulties in analysis and detection of phosphohistidine. Therefore a screening method was developed primarily for the analysis of phosphohistidine phosphatase 1 (PHPT1) activity.

Methods. A highly positively charged substrate, Ac-Val-Arg-Leu-Lys-His-Arg-Lys-Leu-Arg-pNA, containing the peptide surrounding the phosphorylated histidine in ion channel KCa3.1 was chemically phosphorylated using phosphoramidate. Excess phosphoramidate was removed by anion exchange chromatography using a micro spin column. After incubation of the eluate with PHPT1, the removed phosphate was bound on a consecutive anion exchange spin column. The eluate was assayed in a micro plate format for remaining phosphate in the substrate Ac-Val-Arg-Leu-Lys-His(P)-Arg-Lys-Leu-Arg-pNA. Histone H4, also highly positive in charge, was subjected to the same procedure to explore the possibility to use other substrates to PHPT1 in this assay format.

Results. It was found that Ac-Val-Arg-Leu-Lys-His(P)-Arg-Lys-Leu-Arg-pNA and phosphohistone H4 were dephosphorylated by PHPT1. The apparent Km for Ac-Val-Arg-Leu-Lys-His(P)-Arg-Lys-Leu-Arg-pNA was in the order of 10 μM.Using this method, phosphohistidine phosphatase activity was detected in mouse liver cell sap with Ac-Val-Arg-Leu-Lys-His(P)-Arg-Lys-Leu-Arg-pNA as substrate.

Discussion. The described method for determination of PHPT1 activity is comparably much easier and faster than presently used methods for detection of phosphohistidine phosphatase activity. It is also sensitive, since the lower activity limit was 5 pmol phosphate released per min. It has the potential to be used both for more rapid screening for inhibitors and activators to phosphohistidine phosphatases and for screening of histidine kinases.

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Published
2011-06-17
How to Cite
Beckman-Sundh, U., Ek, B., Zetterqvist, Örjan, & Ek, P. (2011). A screening method for phosphohistidine phosphatase 1 activity. Upsala Journal of Medical Sciences, 116(3), 161–168. https://doi.org/10.3109/03009734.2011.585253
Section
Original Articles