Analysis of calcineurin activity by capillary electrophoresis with laser-induced fluorescence detection using peptide substrate.
Reversible protein phosphorylation and dephosphorylation are very important activities in understanding cellular signaling networks. In this paper we described a CE-LIF-based assay method of calcineurin (CN), a protein phosphatase important in cardiac hypertrophy, in which a fluorescence-labeled 19-amino acid phosphopeptide was used as a substrate. The substrate was converted to a dephosphorylated product by CN and both the substrate and product were detected by the LIF detector. This assay method was tested for various separation parameters as well as reaction parameters. It was found that 100 mM of a boric acid buffer with a pH of 9.00 produced optimum separation at 10 kV of applied voltage using a 47 cm capillary. After obtaining the suitable reaction conditions the method was used to detect and quantify the CN activity in HL-1 cell extracts where the picogram level of CN activity was obtained per microgram total protein. It was also observed that immunosuppressive drugs like okadaic acid and cyclosporine A inhibit in vitro CN activity.
Enayetul Babar
Shiekh Md
SM
Bioanalysis and Biotransformation Research Center, Korea Institute of Science and Technology, Seoul, Korea.
Song
Eun Joo
EJ
Yoo
Young Sook
YS
D000595:Amino Acid Sequence; D000818:Animals; D019703:Calcineurin / Q000032:analysis*; D065095:Calcineurin Inhibitors; D002478:Cells, Cultured; D016572:Cyclosporine / Q000737:chemistry / Q000494:pharmacology; D019075:Electrophoresis, Capillary / Q000379:methods*; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology; D005453:Fluorescence; D007834:Lasers; D051379:Mice; D019319:Okadaic Acid / Q000737:chemistry / Q000494:pharmacology; D010748:Phosphopeptides / Q000737:chemistry*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013997:Time Factors
D065095:Calcineurin Inhibitors; D004791:Enzyme Inhibitors; D010748:Phosphopeptides; D019319:Okadaic Acid; D016572:Cyclosporine; D019703:Calcineurin