Department of Medical Biochemistry and Microbiology, Uppsala University, Uppsala, Sweden.
Ups J Med Sci. 2011 Aug;116(3):161-8. doi: 10.3109/03009734.2011.585253. Epub 2011 Jun 17.
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 K(m) 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.
摘要 引言。由于磷酸化组氨酸的分析和检测存在困难，蛋白质结合磷酸组氨酸磷酸化的研究一直受到阻碍。因此，开发了一种主要用于分析磷酸组氨酸磷酸酶 1（PHPT1）活性的筛选方法。 方法。使用磷酰胺将包含在离子通道 KCa3.1 中磷酸化组氨酸周围肽的高度正电荷底物 Ac-Val-Arg-Leu-Lys-His-Arg-Lys-Leu-Arg-pNA 化学磷酸化。通过使用微螺旋柱的阴离子交换层析去除多余的磷酰胺。洗脱液与 PHPT1 孵育后，去除的磷酸结合在连续的阴离子交换螺旋柱上。洗脱液在微孔板格式中测定底物 Ac-Val-Arg-Leu-Lys-His（P）-Arg-Lys-Leu-Arg-pNA 中的剩余磷酸。组蛋白 H4 也带正电荷，也进行了相同的程序，以探索在这种测定形式中使用其他底物对 PHPT1 的可能性。 结果。发现 Ac-Val-Arg-Leu-Lys-His（P）-Arg-Lys-Leu-Arg-pNA 和磷酸化组蛋白 H4 被 PHPT1 去磷酸化。Ac-Val-Arg-Leu-Lys-His（P）-Arg-Lys-Leu-Arg-pNA 的表观 K(m)约为 10 μM。使用该方法，以 Ac-Val-Arg-Leu-Lys-His（P）-Arg-Lys-Leu-Arg-pNA 为底物，在小鼠肝胞浆中检测到磷酸组氨酸磷酸酶活性。 讨论。所描述的 PHPT1 活性测定方法比目前用于检测磷酸组氨酸磷酸酶活性的方法简单快捷得多。它也很灵敏，因为最低活性下限为每分钟释放 5 pmol 磷酸。它有可能既用于更快地筛选磷酸组氨酸磷酸酶的抑制剂和激活剂，也用于筛选组氨酸激酶。
