title: A continuous spectrophotometric assay for mitogen-activated protein kinase kinases.
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 360nm 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.
authors.affiliation: Key Laboratory for Biomechanics and Mechanobiology of Ministry of Education and School of Biological Science and Medical Engineering, Beihang University, Beijing 100191, China.
authors.forename: Li-Sha
authors.initials: LS
authors.lastname: Zheng
authors.forename: Yuan-Yuan
authors.initials: YY
authors.lastname: Zhang
authors.forename: Jia-Wei
authors.initials: JW
authors.lastname: Wu
authors.forename: Zhenguo
authors.initials: Z
authors.lastname: Wu
authors.forename: Zhong-Yin
authors.initials: ZY
authors.lastname: Zhang
authors.forename: Zhi-Xin
authors.initials: ZX
authors.lastname: Wang
mesh_terms: D054642:Dual Specificity Phosphatase 6 / Q000378:metabolism; D054637:Dual-Specificity Phosphatases / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D048369:MAP Kinase Kinase 1 / Q000032:analysis / Q000378:metabolism; D048669:MAP Kinase Kinase 6 / Q000032:analysis / Q000378:metabolism; D020929:Mitogen-Activated Protein Kinase Kinases / Q000032:analysis* / Q000378:metabolism; D054639:Mitogen-Activated Protein Kinase Phosphatases / Q000378:metabolism; D010766:Phosphorylation; D015203:Reproducibility of Results; D013053:Spectrophotometry / Q000379:methods*
chemical_list: D048369:MAP Kinase Kinase 1; D048669:MAP Kinase Kinase 6; C482125:MAP2K1 protein, human; C482566:MAP2K6 protein, human; D020929:Mitogen-Activated Protein Kinase Kinases; C120407:DUSP10 protein, human; D054639:Mitogen-Activated Protein Kinase Phosphatases; C517035:DUSP6 protein, human; D054642:Dual Specificity Phosphatase 6; D054637:Dual-Specificity Phosphatases