A NONRADIOACTIVE FLUORESCENT GEL-SHIFT ASSAY FOR THE ANALYSIS OF PROTEIN PHOSPHATASE AND KINASE-ACTIVITIES TOWARD PROTEIN-SPECIFIC PEPTIDE-SUBSTRATES

Synthetic peptides are important tools with which to study the activit ies of protein kinases and phosphatases toward specific substrate sequ ences which are present within selected regions of a protein. Most exi sting assays for the phosphorylation or dephosphorylation of such pept ides utilize P-32 and either affinity chromatography or HPLC separatio n and require extensive characterization and validation. Here, we desc ribe a method for monitoring the phosphorylation or dephosphorylation of almost any peptide of interest which does not require the use of ra dioactivity, making its reagents stable for a prolonged period, and wh ich can be performed in any standard laboratory. For this, after perfo rmance of kinase or phosphatase reactions with the peptide of interest , products are derivatized with fluorescamine and are separated accord ing to charge by agarose gel electrophoresis. Phosphorylated and nonph osphorylated peptides are readily separated and can be both identified and quantified by uv detection. The lower limit for detection of pept ide in the agarose gel was 0.02 nmol using the gel-shift kinase assay with cAMP-dependent kinase and Kemptide as substrate. This had sensiti vity and reproducibility similar to those of a standard assay using [g amma-P-32]ATP With this substrate. Dephosphorylation of a synthetic ph osphopeptide corresponding to a segment of the cholecystokinin recepto r was tested in an analogous assay with known amounts of protein phosp hatase 2A. Phosphopeptide and dephosphopeptide were easily detected an d quantified with as little as 0.03 mU/mI protein phosphatase 2A activ ity. Therefore, with this assay, most synthetic peptides and phosphope ptides can be used as substrates without further modification. This wi ll be of particular interest for monitoring the purification of highly specific protein kinase and phosphatase activities. (C) 1994 Academic Press, Inc.