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

Douglas E. Wright | Zendy; E.S. Noiman | Zendy; P Boon Chock | Zendy; Vincent Chau | Zendy

Open Access

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

Author(s) -

Douglas E. Wright,

E.S. Noiman,

P Boon Chock,

Vincent Chau

Publication year - 1981

Publication title -

proceedings of the national academy of sciences

Language(s) - English

Resource type - Journals

SCImago Journal Rank - 5.011

H-Index - 771

eISSN - 1091-6490

pISSN - 0027-8424

DOI - 10.1073/pnas.78.10.6048

Subject(s) - phosphoprotein , phosphorylation , phosphatase , peptide , protein kinase a , biochemistry , chemistry , kinase , enzyme kinetics , dephosphorylation , phosphotransferase , adenosine , dusp6 , microbiology and biotechnology , enzyme , biology , protein phosphatase 2 , active site

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 Km and kcat for the phosphorylation reaction at pH 7.0 (25 degrees C), were determined to be 2.7 +/- 0.5 microM 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 Km of 113 +/- 10 microM and a kcat 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 cyclic AMP-dependent protein kinase and phosphoprotein phosphatase activities.

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