All assays are done in a final volume of 50 μL. Each point is tested in duplicate. The standard assay conditions are 50 mM HEPES, 0.2 mM [γ-³²P]ATP (specific activity 200?300 cpm/pmol), 1 mM DTT, 1 mg/mL bovine serum albumin, 5 mM MgCl₂, 150 mM KCl, 15 mM NaCl, pH 7.5, with or without inhibitors. Synthetic substrate concentrations, KKRPQRATSNVFAM-NH2 for MLCK, PLRRTLSVAA-NH2 for CaMPKII, and LRRASLG for PKA, are 20 μM. The reactions are initiated with the addition of 5 μL of enzyme (PKA) or enzyme:CaM complex (for MLCK and CaMPKII). Enzyme:CaM complexes are preformed by incubation of enzyme in ice for 1 min in the presence of 1 μM CaM and 5 μM CaCl₂ (for MLCK) or 30 μM CaM and 2 mM CaCl₂ (for CaMPKII). Assay conditions are 20 min, 25°C for MLCK; 8 min, 25°C for CaMPKII; 6 min, 25°C for PKA. Aliquots of each reaction tube are spotted into phosphocellulose filter paper. Filters are washed in 75 mM phosphoric acid solution, followed by 95% ethanol, dried, and placed into vials containing 10 mL of Ecoscint O. Radioactivity is determined by counting vials in a Beckman 6500 liquid scintillation counter. No quenching corrections are applied. Data are transformed as percent of the maximal enzyme activity, where enzyme activity in the absence of inhibitors is taken as 100%. ICsub>50 values are calculated by a linear regression data analysis on data points corresponding to 15%?85% of maximal activity. This analysis is done using InPlot version 4.0.

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

• [1]. Lukas TJ, et al. Identification of novel classes of protein kinase inhibitors using combinatorial peptide chemistry based on functional genomics knowledge. J Med Chem. 1999 Mar 11;42(5):910-9.  [Content Brief]