Fluorometric assay for adenosine 3',5'-cyclic monophosphate-dependent protein kinase and phosphoprotein phosphatase activities

A novel peptide substrate for adenosine 3',5'-cyclic monophosphate-dependent protein kinase (ATP:protein phosphotransferase, EC 2.7.1.37), Leu-Arg-Arg-Trp-Ser-Leu-Gly, was synthesized. Phosphorylation of the peptide causes a 20% increase in the peptide fluorescence intensity at 358 nm. Values of k(m) and k(cat) for the phosphorylation reaction at pH 7.0 (25°C), were determined to be 2.7 ± 0.5 μM and 5.5 ± 0.4 sec^-1, respectively. The phosphorylated peptide was shown to be an effective substrate for phosphoprotein phosphatase (phosphoprotein phosphohydrolase, EC 3.1.3.16) with a K(m) of 113 ± 10 μM and a k(cat) of 2.4 ± 0.2 sec^-1 in the presence of 2.5 mM MnCl₂. Changes in the peptide fluorescence intensity as a function of its phosphorylation state provide a highly sensitive assay of cylic AMP-dependent protein kinase and phosphoprotein phosphatase activities.