9] Assay of protein kinases using peptides with basic residues for phosphocellulose binding

Abstract

Although various techniques are available to separate the phosphorylated peptide product from other 32 P-labeled material present in assay mixtures, the most efficient method is to make use of the ability of peptides containing several basic charges to bind via ionic interactions to phosphocellulose paper. This technique originated with assays using histones as substrates. Even after phosphorylation, histones are still highly positively charged and bind to phosphocellulose. The pieces of phosphocellulose can be washed in water and the phosphorylated histones are retained while the [ γ - 32 P]ATP and any [ 32 P]P i are washed away. As some peptide substrates contain only two basic residues, it is necessary to modify the assay by using acid washes that suppress the negative charges of the phosphate group. The phosphocellulose technique has been extended to enzymes that recognize acidic residues in their peptide substrates instead of basic residues. Although the phosphocellulose assay can be used with peptide substrates that have only a single basic residue, quantitative retention on the phosphocellulose paper after phosphorylation requires the presence of at least two basic residues and a free amino terminus. The phosphocellulose assay is straightforward and highly sensitive. The only problem that is encountered with the assay is high blanks. Even when assaying for activity in crude extracts the blanks should be 0.1% or less of the total input counts.