Phosphorylation of acyl and dansyl derivatives of the peptide Leu-Arg-Arg-Ala-Ser-Leu-Gly by the cAMP-dependent protein kinase.

A series of acyl derivatives and a dansyl derivative of the synthetic peptide Leu-Arg-Arg-Ala-Ser-Leu-Gly have been tested as substrates for the catalytic subunit of the CAMP-dependent protein kinase (ED 2.7.1.37; ATP: phosphotransferase). The purpose of this study was to test whether substitution of the NH2-terminal leucine with acyl substituents of varying chain length and hydrophobicity would influence the kinetics of phosphorylation of these peptides. Increasing the acyl substituent from acetyl to hexanoyl had no marked effect on the kinetics of phosphorylation. The trifluoroacetyl derivative was also phosphorylated with kinetic parameters comparable to the parent peptide, namely an apparent K , of 2.5 ~ L M and a v, of 30 pmolmin” mg”. Substitution of the pentapeptide Arg-AlaSer-Leu-Gly with acyl substituents also had little effect on the kinetics of phosphorylation. The a-N dansyl derivative of Leu-Arg-Arg-Ala-Ser-Leu-Gly-COOH was phosphorylated by the protein kinase with an apparent K,,, of 3 pM and a V,, of 27 pmol. min” emg“. The dansyl peptide appeared to act as a specific substrate for the cyclic AMP-dependent protein kinase as indicated by DE-52 ion exchange chromatography of crude extracts of rat muscle. Since the phosphorylated form of the dansyl peptide was readily separated from the dephospho form by high pressure liquid chromatography on a reverse phase octadecasilane column the protein kinase could be assayed spectrophotometrically using this technique.