A Novel and Simple Method to Assay the Activity of Individual Protein Kinases in a Crude Tissue Extract

✍ Scribed by B.S. Goueli; K. Hsiao; A. Tereba; S.A. Goueli

• Publisher: Elsevier Science
• Year: 1995
• Tongue: English
• Weight: 671 KB
• Volume: 225
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1006/abio.1995.1100

✦ Synopsis

Protein kinases and phosphatases play an important role in a variety of cellular functions. Thus, it is of interest to develop an assay system that can be used to quantify the activity of individual enzymes specifically in a crude cellular extract, is simple to perform, and is amenable to automation. Here we report on the development of a protein kinase assay that addresses these points and circumvents the pitfalls of 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 (\left[\gamma-{ }^{32}\right.) P]ATP under optimal enzyme condition, and the phosphorylated peptide product is then captured by a streptavidin-linked disk. After removal of free ([\gamma-) ({ }^{32} P) JATP, the ({ }^{32} P) incorporated into the peptide substrate can be used as an expression of enzyme activity. In contrast to the commonly used phosphocellulose method, only the phospho-, biotinylated peptide (and not other phosphorylated proteins present in the extract) will bind to the disks, thus giving a true estimate of enzymatic activity. In addition to specificity, this assay does not require the peptide substrate to contain basic amino acids or to be modified by the addition of basic amino acid residues as required for the phosphocellulose method which may result in altered 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 (\mathrm{pH}), temperature, ionic and nonionic detergents, organic solvents, and other denaturing agents. 1995 Academic Press, Inc.