A generic medium throughput activity assay procedure for serine–threonine protein phosphatases using 14C-labelled N-acetyl-Arg-Arg-Ala-Thr(P)-Val-Ala

Abstract

A new and sensitive activity assay for the Ser-Thr protein phosphatases PP1 and PP2A based upon the hydrolysis of the synthetic radiolabelled phosphopeptide substrate N-acetyl-Arg-Arg-Ala-Thr(P)-Val-Ala, is described. The protocol is also applicable to the assay of and PP2C activity. The radiolabelled phosphopeptide is stable and can be stored for prolonged periods without deterioration or loss of radioactivity offering advantages over the use of ³²P-labelled substrates. The assay method involves the separation of phosphopeptide substrate from the peptide alcohol product by anion exchange chromatography. The separation protocol is not sensitive to the presence of inorganic phosphate anion or metal cations. Using PP1 as the enzyme the radiochemical assay procedure afforded a V_max value of 17 (±2) μM s⁻¹ [28 (±3) μM s⁻¹ μg⁻¹] and a K_M value of 3.7 (±0.9) mM for the substrate Ac-Arg-Arg-Ala-Thr(P)-Val-Ala with significantly greater accuracy than for a Malachite Green based assay. Inorganic phosphate was shown to be a competitive product inhibitor (K_i = 1.6 mM) and nodularin was found to be a potent competitive inhibitor (K_i = 0.19 nM) for PP1 with respect to the phosphopeptide substrate. This assay procedure was employed to determine the mode and magnitude of the inhibition of PP1 by the nodularin analogue described in the previous article [M. E. O'Donnell, J. Sanvoisin and D. Gani, J. Chem. Soc., Perkin Trans. 1, 2001 (DOI:10.1039/b100402f)]. The enzyme PP2A afforded a V_max value of 2.8 (±0.2) μM s⁻¹ [45 (±2) μM s⁻¹μg⁻¹] and a K_M value of 4.1 (±0.7) mM for the radiolabelled substrate.