The following two fluorescent peptides were prepared as substrates of the cyclic AMP-dependent protein kinase reaction: Leu–Arg–Arg–Ala–Ser–Leu–X; 1 (X= 2-(dansylamino)ethylamino or DAE) and 2 (X=4-methyl-2-oxo-7-chromenylamino or MCA). Both peptides were phosphorylated by ATP and the catalytic subunit of cyclidAMP-dependent protein kinase from bovine heart in a stoichiometric manner. Phosphorylation of peptide 1 is accompanied by a 10% increase in the fluorescence intensity at 550 nm. This enables one to assay peptide phosphorylation by fluorescence spectroscopy. The kinetic parameters obtained by this and the conventional phosphocellulose paper method for this phosphorylation were the following: Km 7.8 μM and Vmax 35 μmol·min−1·mg−1 for 1, and Km 7.4μM and Vmax 16 μmol·min−1·mg−1 for 2.
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