A CAPILLARY ELECTROPHORESIS-BASED ASSAY FOR PROTEIN-KINASES AND PROTEIN PHOSPHATASES USING PEPTIDE-SUBSTRATES

A novel procedure for detection and assay of protein kinase and phosph atase activities in complex biological mixtures was developed. By mean s of capillary zone electrophoresis (CZE) methodology, the phosphoryla ted and dephosphorylated forms of the peptide Kemptide, a 46-amino-aci d fragment from protein phosphatase inhibitor-1 and a peptide fragment corresponding to the R(II) subunit of cAMP-dependent protein kinase ( PKA), were rapidly resolved. This facilitated nonradioactive detection of PKA and protein phosphatase-2B (calcineurin) in rabbit skeletal mu scle extracts. In addition, the CZE procedure enabled a site-specific assay of a 14-amino-acid peptide from the glycogen-binding subunit of protein phosphatase-1 monophosphorylated on distinct sites by PKA and casein kinase-II. These results suggest that CZE may prove to be extre mely useful for the analysis of peptides that are phosphorylated at mu ltiple sites in vivo. (C) 1994 Academic Press, Inc.