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- Beckman-Sundh, UllaUppsala universitet,Institutionen för medicinsk biokemi och mikrobiologi (author)
A screening method for phosphohistidine phosphatase 1 activity
- Article/chapterEnglish2011
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- 2011-06-17
- Uppsala Medical Society,2011
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- LIBRIS-ID:oai:DiVA.org:uu-156940
- https://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-156940URI
- https://doi.org/10.3109/03009734.2011.585253DOI
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- Language:English
- Summary in:English
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- 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 K(m) for Ac-Val-Arg-Leu-Lys-His(P)-Arg-Lys-Leu-Arg-pNA was in the order of 10 mu M. 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.
Subject headings and genre
- MEDICIN OCH HÄLSOVETENSKAP Medicinska och farmaceutiska grundvetenskaper Mikrobiologi inom det medicinska området hsv//swe
- MEDICAL AND HEALTH SCIENCES Basic Medicine Microbiology in the medical area hsv//eng
- Activity
- assay
- phosphohistidine phosphatase
- PHPT1
- protein histidine phosphatase
- Medical microbiology
- Medicinsk mikrobiologi
Added entries (persons, corporate bodies, meetings, titles ...)
- Ek, BoUppsala universitet,Analytisk kemi,Institutionen för medicinsk biokemi och mikrobiologi,Science for Life Laboratory, SciLifeLab(Swepub:uu)boaek194 (author)
- Zetterqvist, ÖrjanUppsala universitet,Institutionen för medicinsk biokemi och mikrobiologi(Swepub:uu)oze13294 (author)
- Ek, PiaUppsala universitet,Institutionen för medicinsk biokemi och mikrobiologi(Swepub:uu)pek21046 (author)
- Uppsala universitetInstitutionen för medicinsk biokemi och mikrobiologi (creator_code:org_t)
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- In:Upsala Journal of Medical Sciences: Uppsala Medical Society116:3, s. 161-1680300-97342000-1967
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