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Fluram-Kemptide-Lys8 Non-radioactive Assay for Protein Kinase A

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Abstract

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. Measurements of PKA activity traditionally relied on the use of [32P]-labeled ATP as the phosphate donor and a protein or peptide substrate as the phosphoaceptor. 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. 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/TΨ), where X is any amino acid and Ψ is a hydrophobic amino acid. 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. The phosphorylated and non-phosphorylated peptides were easily separated by electrophoresis, identified and quantified with optical densitometry and ultraviolet light. 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 700 and 1150 pmol with a linear regression R 2 = 0.956.

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Abbreviations

ATP:

Adenosine triphosphate

AMP:

Adenosine monophosphate

cAMP:

Cyclic adenosine monophosphate

PKA:

Protein kinase A

PKAc:

Catalytic subunit of protein kinase A

UV:

Ultraviolet

HPLC:

High-performance liquid chromatography

Km:

Michaelis–Menten constant

Fluram:

Fluorescamine

Lys:

Lysine

Fluram-Kemptide-Lys8:

Fluorescamine-labeled Kemptide-Lys8

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Acknowledgments

This research was supported by Grant No. 2013001659 from FONACIT, Venezuela. We want to thank Dr. Oscar Noya (Universidad Central de Venezuela) for synthesizing and donating the peptides that were used in this work.

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Authors and Affiliations

  1. Departamento de Biología Celular, División de Ciencias Biológicas, Universidad Simón Bolívar, Apartado 89.000, Valle de Sartenejas, Baruta, Caracas, 1081, Venezuela

    Nelson A. Araujo, Alberto Guevara, Maritza Calabokis & José Bubis

  2. Postgrado en Química, Departamento de Química, Universidad Simón Bolívar, Caracas, Venezuela

    Nelson A. Araujo

  3. Postgrado en Ciencias Biológicas, Departamento de Biología Celular, Universidad Simón Bolívar, Caracas, Venezuela

    Alberto Guevara

  4. Sección de Biohelmintiasis, Instituto de Medicina Tropical, Escuela de Medicina “Luis Razetti”, Facultad de Medicina, Universidad Central de Venezuela, Caracas, Venezuela

    María A. Lorenzo

Authors
  1. Nelson A. Araujo
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  2. Alberto Guevara
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  3. María A. Lorenzo
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  4. Maritza Calabokis
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  5. José Bubis
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Corresponding author

Correspondence to Nelson A. Araujo.

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Araujo, N.A., Guevara, A., Lorenzo, M.A. et al. Fluram-Kemptide-Lys8 Non-radioactive Assay for Protein Kinase A. Protein J 35, 247–255 (2016). https://doi.org/10.1007/s10930-016-9667-9

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