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

D. E. Wright, E. Shacter Noiman, P. Boon Chock, V. Chau

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Abstract

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.

Original languageEnglish (US)
Pages (from-to)6048-6050
Number of pages3
JournalProceedings of the National Academy of Sciences of the United States of America
Volume78
Issue number10 I
DOIs
StatePublished - Jan 1 1981

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Phosphoprotein Phosphatases
Cyclic AMP
Protein Kinases
Peptides
Phosphorylation
Fluorescence
Adenosine Monophosphate

All Science Journal Classification (ASJC) codes

  • General

Cite this

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title = "Fluorometric assay for adenosine 3',5'-cyclic monophosphate-dependent protein kinase and phosphoprotein phosphatase activities",
abstract = "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.",
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Fluorometric assay for adenosine 3',5'-cyclic monophosphate-dependent protein kinase and phosphoprotein phosphatase activities. / Wright, D. E.; Shacter Noiman, E.; Boon Chock, P.; Chau, V.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 78, No. 10 I, 01.01.1981, p. 6048-6050.

Research output: Contribution to journalArticle

TY - JOUR

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AU - Wright, D. E.

AU - Shacter Noiman, E.

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AU - Chau, V.

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N2 - 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.

AB - 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.

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