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  • cAMP-dependent Protein Kinase (PKA), catalytic subunit

    Description

    The catalytic subunit of cAMP-dependent Protein Kinase (PKA) is a serine/threonine protein kinase, which combines, in the absence of cAMP, with the regulatory subunit to form the inactive PKA holoenzyme. Since this is the free catalytic subunit alone, no cAMP is required for activation (1,2). 

    When purified from mammalian tissue, the PKA catalytic subunit is always phosphorylated at T197, essential for catalysis. Most likely a heterologous kinase is responsible for this in vivo phosphorylation of PKA. Although the fully active PKA expressed in E. coli autophosphorylates on both T197 and S338, this does not reflect the mechanism used in eukaryotic cells (3). 

    Product Source

    Isolated from a strain of E. coli that carries a clone expressing the murine PKA catalytic subunit (α isoform) under control of a T7 expression system (2,3) (cDNA kindly provided by Dr. G.S. McKnight).

    Recognition Determinant

    The recognition motif for phosphorylation by PKA is RRXS/TY, where Y tends to be a hydrophobic residue. A Phe in the nearby sequence tends to be a negative determinant for phosphorylation by PKA. Some variations with regard to spacing and basic residues are permissible (2,4).

    Reagents Supplied

    The following reagents are supplied with this product:

    Store at (°C)Concentration
    PKA Reaction Buffer-2010X

    Properties and Usage

    Unit Definition

    One unit is defined as the amount of PKA catalytic subunit required to catalyze the transfer of 1 pmol of phosphate to Kemptide, LRRASLG (100 µM) in 1 minute at 30°C in a total reaction volume of 25μL.

    Reaction Conditions

    1X PKA Reaction Buffer
    Supplement with 200 μM ATP
    Incubate at 30°C

    1X PKA Reaction Buffer:
    50 mM Tris-HCl
    10 mM MgCl2
    pH 7.5 @ 25°C

    Storage Temperature

    -20°C

    Storage Conditions

    20 mM Tris-HCl
    50 mM NaCl
    2 mM DTT
    1 mM EDTA
    50% Glycerol
    pH 7.5 @ 25°C

    Heat Inactivation

    65°C for 20 min

    Molecular Weight

    Theoretical: 38 kDa

    Specific Activity

    5,000,000 units/mg

    Quality Control

    Quality Control Assays

    The following Quality Control Tests are performed on each new lot and meet the specifications designated for the product. Individual lot data can be found on the Product Summary Sheet/Datacard or Manual which can be found in the Supporting Documents section of this page.
    • Phosphatase Activity (PNPP):
      The product is tested in a reaction containing a p-nitrophenyl phosphate (PNPP), a chromogenic substrate for most phosphatases. After incubation there is no detectable phosphatase activity as determined by spectrophotometric analysis of released p-nitrophenol at 405 nm.
    • Protease Activity (SDS-PAGE):

      The product is tested for protease activity by incubation with a standard mixture of proteins resulting in no detectable degradation of the proteins as determined by SDS-PAGE.

    Notes

    1. Reaction Conditions: 1X PKA Reaction Buffer, supplemented with 200 µM ATP and gamma-labeled ATP to a final specific activity of 100-500 µCi/μmol.
    2. Optimal incubation times and enzyme concentrations must be determined empirically for each particular substrate.
    3. If possible, the ATP concentration should be at or near saturation (5 -10-fold over Km). Apparent Km values of ATP for most protein kinases are below 100 μM.

      However, if the objective is to measure enzyme activity using gamma-labeled ATP, it is best to use 100-200 μM ATP in order to have higher specific activity of gamma-labeled ATP (100-500 cpm/pmol). Also, an excess of substrate should be used, and the level of phosphorylation should not exceed 10% for determination of the initial rate.

      To phosphorylate a protein or peptide substrate to completion, the ATP concentration should be about 5-fold over the limited substrate concentration. Higher enzyme concentration and prolonged incubation times should be employed.

      Recommended reference:
      Protein Phosphorylation: A Practical Approach (1993) ed. Hardie, D.G. IRL Press.
    4. This clone has a nucleotide sequence identical to the GenBank entry NM_008854, as entered by Dr. G. S. McKnight.
    5. If the source of protein to be phosphorylated is a crude extract of cells or tissue, it is very important to include the appropriate protease and protein phosphatase inhibitors in the lysis buffer and to use shorter incubation time for phosphorylation.

    References

    1. Uhler, M.D., Carmichael, D.F., Lee, D.C., Chrivia, J.C., Krebs, E.G. and McKnight, G.S. (1986). Proc. Natl. Acad. Sci. USA. 83, 1300-1304.
    2. Slice, L.W. and Taylor, S.S. (1989). J. Biol. Chem. 264, 20940-20946.
    3. Moore, M.J. et al. (2002). J. Biol. Chem. 277, 47878-47884.
    4. Zetterqvist, O.Z. et al. (1990). B.E. Kemp, ed(Ed.), in Peptides and Protein Phosphorylation. 171-187. CRC Press, Inc. Boca Raton.

    Supporting Documents

    Material Safety Datasheets

    The following is a list of Material Safety Data Sheets (MSDS) that apply to this product to help you use it safely. The following file naming structure is used to name these document files: [Product Name] MSDS. For international versions please contact us at info@neb.com.

    Datacards

    The Product Summary Sheet, or Data Card, includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name the majority of these document files: [Catalog Number]Datasheet-Lot[Lot Number]. For those product lots not listed below, please contact NEB at info@neb.com or fill out the Technical Support Form for appropriate document.
    1. How much cAMP-dependent Protein Kinase (NEB# P6000) should be used?
    2. What is the consensus sequence for PKA (P6000)?

    Selection Tools

    Make sure to use the appropriate protease and protein phosphatase inhibitors in the lysis buffer and to use shorter incubation time for phosphorylation if the substrate is a crude lysate.
    If possible, the ATP concentration should be at or near saturation (5 -10-fold over Km). Apparent Km values of ATP for most protein kinases are below 100 µM.
    1-2 µl of enzyme is a good starting point for a 1 hr incubation of 1µg of protein.
    The consensus sequence is RRXS/TY, where Y tends to be a hydrophobic residue