A NOVEL AND SIMPLE METHOD TO ASSAY THE ACTIVITY OF INDIVIDUAL PROTEIN-KINASES IN A CRUDE TISSUE-EXTRACT

Protein kinases and phosphatases play an important role in a variety o f cellular functions. Thus, it is of interest to develop an assay syst em that can be used to quantify the activity of individual enzymes spe cifically in a crude cellular extract, is simple to perform, and is am enable to automation. Here we report on the development of a protein k inase assay that addresses these points and circumvents the pitfalls o f existing methodologies. The assay is based on the high affinity and strong binding of streptavidin toward biotin-linked peptide substrates . The biotinylated peptide substrate is phosphorylated by the cognate protein kinase using [gamma-P-32]ATP under optimal enzyme condition, a nd the phosphorylated peptide product is then captured by a streptavid in-linked disk. After removal of free [gamma(-)(32)P]ATP, the P-32 inc orporated into the peptide substrate can be used as an expression of e nzyme activity. In contrast to the commonly used phosphocellulose meth od, only the phospho-, biotinylated peptide (and not other phosphoryla ted proteins present in the extract) will bind to the disks, thus givi ng a true estimate of enzymatic activity. In addition to specificity, this assay does not require the peptide substrate to contain basic ami no acids or to be modified by the addition of basic amino acid residue s as required for the phosphocellulose method which may result in alte red specificity of the substrate. Finally, the binding of the modified peptide substrate to the disks is of high affinity, rapid, and, once formed, unaffected by a wide extreme of pH, temperature, ionic and non ionic detergents, organic solvents, and other denaturing agents. (C) 1 995 Academic Press,Inc.