Is the glycogen synthase analogue C1-peptide a suitable fluorescent substrate for routine measurements of protein kinase C?
We have compared a new commercially available non-radioactive protein kinase C (PKC) activity assay based on the fluorescent [A9,10K11]glycogen synthase1-11 analogue C1-peptide with a classical radioactive assay based on myelin basic protein4-14 (MBP4-14) and other substrates. The C1-peptide had lower affinity for PKC from rat brain than substrates such as MBP4-14, [S25]PKC alpha 19-31, and [A9,10K11,12]glycogen synthase1-12. The sensitivity of the C1-peptide-based assay was considerably lower than that of the MBP4-14-based assay. The C1-peptide was readily degraded in an ATP-independent manner by crude and DEAE-column chromatography-purified cytosolic extracts from rat brain, rat kidney, SK-N-MC and L929 cells. In rat kidney this degradation was not prevented by many common protease inhibitors. Phenylsepharose column chromatography separated the C1-peptide degrading activity from PKC. We conclude that the C1-peptide-based fluorescent PKC assay is applicable to highly purified PKC preparations but has low sensitivity and is not applicable to crude extracts due to substrate degradation.
Erdbrügger
W
W
Department of Medicine, University of Essen, Germany.
Strohm
P
P
Michel
M C
MC
D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000201:enzymology; D002458:Cell Fractionation; D005456:Fluorescent Dyes / Q000378:metabolism*; D006006:Glycogen Synthase / Q000378:metabolism*; D007668:Kidney / Q000201:enzymology; D007739:L Cells; D008297:Male; D051379:Mice; D008969:Molecular Sequence Data; D004676:Myelin Basic Protein / Q000378:metabolism; D009842:Oligopeptides / Q000378:metabolism; D010446:Peptide Fragments / Q000378:metabolism*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000302:isolation & purification / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D011933:Reagent Kits, Diagnostic; D012680:Sensitivity and Specificity; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology; D013379:Substrate Specificity; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014407:Tumor Cells, Cultured
C098228:C1 peptide; D005456:Fluorescent Dyes; D004676:Myelin Basic Protein; D009842:Oligopeptides; D010446:Peptide Fragments; D011933:Reagent Kits, Diagnostic; D015842:Serine Proteinase Inhibitors; D006006:Glycogen Synthase; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate