Fluram-Kemptide-Lys8 Non-radioactive Assay for Protein Kinase A.
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 [(32)P]-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.
Departamento de Biología Celular, División de Ciencias Biológicas, Universidad Simón Bolívar, Apartado 89.000, Valle de Sartenejas, Baruta, Caracas, 1081, Venezuela. nelsonaaa@gmail.com.|Postgrado en Química, Departamento de Química, Universidad Simón Bolívar, Caracas, Venezuela. nelsonaaa@gmail.com.
Nelson A
NA
Araujo
Departamento de Biología Celular, División de Ciencias Biológicas, Universidad Simón Bolívar, Apartado 89.000, Valle de Sartenejas, Baruta, Caracas, 1081, Venezuela.|Postgrado en Ciencias Biológicas, Departamento de Biología Celular, Universidad Simón Bolívar, Caracas, Venezuela.
Alberto
A
Guevara
Sección de Biohelmintiasis, Instituto de Medicina Tropical, Escuela de Medicina "Luis Razetti", Facultad de Medicina, Universidad Central de Venezuela, Caracas, Venezuela.
María A
MA
Lorenzo
Departamento de Biología Celular, División de Ciencias Biológicas, Universidad Simón Bolívar, Apartado 89.000, Valle de Sartenejas, Baruta, Caracas, 1081, Venezuela.
Maritza
M
Calabokis
Departamento de Biología Celular, División de Ciencias Biológicas, Universidad Simón Bolívar, Apartado 89.000, Valle de Sartenejas, Baruta, Caracas, 1081, Venezuela.
José
J
Bubis
D000595:Amino Acid Sequence; D000818:Animals; D020134:Catalytic Domain; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry* / Q000302:isolation & purification; D057075:Enzyme Assays / Q000379:methods*; D005450:Fluorescamine / Q000737:chemistry*; D007700:Kinetics; D009206:Myocardium / Q000737:chemistry; D009842:Oligopeptides / Q000138:chemical synthesis / Q000737:chemistry*; D010766:Phosphorylation; D013379:Substrate Specificity; D013552:Swine
D009842:Oligopeptides; D005450:Fluorescamine; C037642:kemptide; D017868:Cyclic AMP-Dependent Protein Kinases
Assay non-radioactive; Fluorescamine; Fluram; Kemptide-Lys8; Protein kinase A