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 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 [gamma-32P]ATP under optimal enzyme condition, and the phosphorylated peptide product is then captured by a streptavidin-linked disk. After removal of free [gamma-32P]ATP, the 32P 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Goueli
B S
BS
Department of Signal Transduction, Promega Corporation, Madison, Wisconsin 53711, USA.
Hsiao
K
K
Tereba
A
A
Goueli
S A
SA
D000818:Animals; D001710:Biotin; D001921:Brain / Q000201:enzymology*; D002417:Cattle; D002845:Chromatography / Q000379:methods; D002848:Chromatography, DEAE-Cellulose / Q000379:methods; D002852:Chromatography, Ion Exchange / Q000379:methods; D017868:Cyclic AMP-Dependent Protein Kinases / Q000032:analysis / Q000302:isolation & purification; D017886:Durapatite; D005260:Female; D007202:Indicators and Reagents; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D009206:Myocardium / Q000201:enzymology*; D009928:Organ Specificity; D010053:Ovary / Q000201:enzymology; D011493:Protein Kinase C / Q000032:analysis / Q000302:isolation & purification; D011494:Protein Kinases / Q000032:analysis* / Q000302:isolation & purification / Q000378:metabolism*
D007202:Indicators and Reagents; D001710:Biotin; D017886:Durapatite; D011494:Protein Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C