PMID: 8588980Aug 1, 1995Paper

Is the glycogen synthase analogue C1-peptide a suitable fluorescent substrate for routine measurements of protein kinase C?

Cellular Signalling
W ErdbrüggerM C Michel

Abstract

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.

References

Feb 14, 1990·Biochemical and Biophysical Research Communications·I YasudaY Nishizuka
Oct 31, 1991·Biochemical and Biophysical Research Communications·Y J FarrarJ T Slevin
May 15, 1991·Analytical Biochemistry·B K McIlroyJ D Johnson
Feb 1, 1994·Trends in Biochemical Sciences·L V Dekker, P J Parker

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