Measuring Protein Phosphatase Activity with Physiological Substrates

Abstract

This chapter discusses Protein phosphorylation as a major posttranslational modification mechanism that a cell utilizes to control its biological processes. Protein phosphatases are hydrolytic enzymes that remove the phosphoryl group from the phosphorylated residue(s). The chapter outlines three assays that study the dephosphorylation of the classical MAP kinase ERK2 by protein phosphatases—namely, radioactive assay, enzyme-coupled continuous spectrophotometric assay—and determining the activity of a protein phosphatase from its effect on a kinase reaction. In principal, these assays are also suited for detailed biochemical analysis of protein phosphatases with other protein substrates. In radioactive assay, protein phosphatase activity is determined from the release of 32 P inorganic phosphate (Pi) from the 32 P-labeled ERK2. This is one of the most commonly used assays for protein phosphatases because of its high sensitivity. Enzyme-coupled continuous spectrophotometric assay is a nonradioactive, continuous spectrophotometric assay for monitoring protein dephosphorylation. In this coupled enzyme system, the coupling enzyme, purine nucleoside phosphorylase, uses the inorganic phosphate, generated by the action of the phosphatase, to convert 7-methyl-6-thioguanosine to 7-methyl-6-thioguanine and ribose-1-phosphate, resulting in an increase in absorbance at 360 nm.