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cAMP-dependent Protein Kinase (PKA), catalytic subunit |
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- Protein serine/threonine kinase
- Mouse, recombinant (E. coli)
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).
Recognition Determinants:
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).
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).
Reagents Supplied:
PKA Reaction Buffer (10X)
Enzyme Properties
Protein Kinase Substrate Recognition
Heat Inactivation:
65°C for 20 minutes
Molecular Weight:
Theoretical: 38 kDa
Specific Activity:
5,000,000 units/mg
Reaction & Storage Conditions
Reaction Conditions:
1X PKA Reaction Buffer
Supplemented with 200 μM ATP and 300 μCi/μmol gamma-labeled ATP
Incubate at
30°C.
1X PKA Reaction Buffer:
50 mM Tris-HCl
10 mM MgCl2
pH 7.5 @ 25°C
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, NEB #P6001) in 1 minute at 30°C in a total reaction volume of 25 µl.
Concentration:
2,500,000 units/ml
Storage Conditions:
20 mM Tris-HCl
50 mM NaCl
2 mM DTT
1 mM EDTA
50% Glycerol
pH 7.5 @ 25°C
Storage Temperature:
-20°C
Notes
General notes:- This clone has a nucleotide sequence identical to the GenBank entry NM_008854, as entered by Dr. G. S. McKnight.
- 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.
- 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.
Usage notes:- Optimal incubation times and enzyme concentrations must be determined empirically for each particular substrate.
- 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.
FAQs
- How much cAMP-dependent Protein Kinase (NEB# P6000) should be used?
- What is the consensus sequence for PKA (P6000)?
Quality Control for Current Lot
Quality control values for a specific lot can be found on the datacard which accompanies each product.
Quality Assurance Statement:
PKA contains no detectable protease or phosphatase activities.
Protease Activity:
After incubation of 20,000 units of cAMP-dependent Protein Kinase (PKA), catalytic subunit with a standard mixture of proteins
for 2 hours at 30ºC, no proteolytic activity could be detected by SDS-PAGE.
Contaminating Phosphatases:
After incubation of 20,000 units of with 50 mM p-nitrophenyl phosphate for 2 hours at 30ºC, no
phosphatase activity could be detected by spectrophotometric analysis.
References
- 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.
- Slice, L.W. and Taylor, S.S. (1989) J. Biol. Chem., 264, 20940-20946.
- Moore, M.J. et al. (2002) J. Biol. Chem., 277, 47878-47884.
- Zetterqvist, O.Z. et al. (1990) in Peptides and Protein Phosphorylation, (B.E. Kemp, ed), pp. 171-187, CRC Press, Inc., Boca Raton.
Reagents Sold Separately
PKA Reaction Buffer
Companion Products
Adenosine 5´-Triphosphate (ATP)
Kemptide (PKA Peptide Substrate)
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