A continuous spectrophotometric assay for mitogen-activated protein kinase kinases

Li Sha Zheng, Yuan Yuan Zhang, Jia Wei Wu, Zhenguo Wu, Zhong Yin Zhang, Zhi Xin Wang

Research output: Contribution to journalArticle

5 Scopus citations

Abstract

We describe a convenient and simple continuous spectrophotometric method for the determination of mitogen-activated protein kinase (MAPK) kinase activity with its protein substrate. The assay relies on the measurement of phosphoprotein product generated in the first step of the MAPK kinase reaction. Dephosphorylation of the phosphoprotein is coupled to a MAPK phosphatase to generate phosphate, which is then used as the substrate of purine nucleoside phosphorylase to catalyze the N-glycosidic cleavage of 2-amino 6-mercapto 7-methyl purine ribonucleoside. Of the reaction products ribose 1-phosphate and 2-amino 6-mercapto 7-methylpurine, the latter has a high absorbance at 360 nm relative to the nucleoside and, hence, provides a spectrophotometric signal that can be continuously followed. In the presence of excess phosphatase, the phosphorylated protein substrate molecules undergo dephosphorylation almost immediately after their formation; the steady-state use of the resultant inorganic phosphate is a reflection of the constant initial velocity of the exchange reaction. The validity of this method has been confirmed by using it to measure the activities of MEK1 (MAPK/ERK kinase 1) and MKK6 (MAPK kinase 6) toward their physiological substrates. Our findings of the MAPK kinases in the current study provide evidence that the substrate binding affinities of this subfamily of protein kinases are at the submicromolar concentration.

Original languageEnglish (US)
Pages (from-to)191-197
Number of pages7
JournalAnalytical biochemistry
Volume421
Issue number1
DOIs
StatePublished - Feb 1 2012

    Fingerprint

Keywords

  • Dephosphorylation
  • MAP kinase
  • MAP kinase kinase
  • MAP kinase phosphatase
  • Phosphorylation

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology
  • Cell Biology

Cite this