![]() The fluorescamine-labeled Kemptide-Lys8 substrate (Fluram-Kemptide-Lys8) was used to calculate the Km and Vmax of the catalytic subunit of PKA from pig heart and showed a detection limit of 260 pmol, a linear range between 7 pmol with a linear regression R2 = 0.956. The phosphorylated and non-phosphorylated peptides were easily separated by electrophoresis, identified and quantified with optical densitometry and ultraviolet light. The cyclic AMP assay is a functional assay that is commonly used to determine the pharmacological behavior (agonists, antagonists, and inverse agonists) of G-protein coupled receptor ligands. Optimal conditions were established for the non-radioactive assay to detect the PKA activity by phosphorylation of these three peptides that are covalently linked to fluorescamine at their N-terminus. Three peptides were synthesized with the following sequences: LRRASLG (Kemptide), LRRASLGK (Kemptide-Lys8) and LRRASLGGGLRRASLG (Bis-Kemptide), these have in common the substrate sequence recognized by the PKA (RRXS/TW), where X is any amino acid and W is a hydrophobic amino acid. Recently non-isotopic assays for the PKA have been developed and this paper presents an improvement of a fluorometric assay for measuring the activity of PKA. Measurements of PKA activity traditionally relied on the use of -labeled ATP as the phosphate donor and a protein or peptide substrate as the phosphoaceptor. The cAMP-dependent protein kinase (PKA) is the best understood member of the superfamily of serine–threonine protein kinases and is involved in controlling a variety of cellular processes.
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