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 phosphatase activities in complex biological mixtures was developed. By means of capillary zone electrophoresis (CZE) methodology, the phosphorylated and dephosphorylated forms of the peptide Kemptide, a 46-amino-acid fragment from protein phosphatase inhibitor-1 and a peptide fragment corresponding to the RII subunit of cAMP-dependent protein kinase (PKA), were rapidly resolved. This facilitated nonradioactive detection of PKA and protein phosphatase-2B (calcineurin) in rabbit skeletal muscle 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 extremely useful for the analysis of peptides that are phosphorylated at multiple sites in vivo.
Dawson
J F
JF
Department of Biochemistry, University of Alberta, Edmonton, Canada.
Boland
M P
MP
Holmes
C F
CF
D000818:Animals; D002197:Capillary Action; D017868:Cyclic AMP-Dependent Protein Kinases / Q000032:analysis; D004586:Electrophoresis / Q000379:methods; D007700:Kinetics; D009132:Muscles / Q000201:enzymology*; D009842:Oligopeptides / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism*; D010749:Phosphoprotein Phosphatases / Q000032:analysis* / Q000378:metabolism; D010766:Phosphorylation; D011494:Protein Kinases / Q000032:analysis* / Q000378:metabolism; D054645:Protein Phosphatase 1; D011817:Rabbits; D013379:Substrate Specificity
D009842:Oligopeptides; D010446:Peptide Fragments; C037642:kemptide; D011494:Protein Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1