Kmvalue for the specific isoform and to a suggested final^33 P-
ATP specific activity of 0.25μCi/nmol (seeNote 40). Since
this solution will be diluted 4when added to the reaction, the
final (ATP) in the reaction will be 10the ATPKmvalue. 4
SAMS peptide substrate mixture—Prepare SAMS peptide solu-
tion by diluting SAMS peptide in 1kinase buffer to a con-
centration equal to 40the reportedKmvalue for the specific
isoform. Serially dilute the SAMS mix to cover a concentration
range of 0.1 to 10-fold the expectedKm value in the final
reaction mixture.
- Add 10μl of the activator mixture and 10μl of the AMPK
enzyme mixture to a 384-well polypropylene plate. Incubate
the plate at room temperature for 15 min to allow enzyme and
compound to come to equilibrium.
- Add 10μl of the ATP mixture.
- Add 10μl of the SAMS peptide substrate mixture to initiate the
kinase reaction (seeNote 41). Reaction time courses can be
performed by initiating reactions in specific wells at different
times and terminating all reactions simultaneously with the
addition of 20μl of 2% (v/v) phosphoric acid.
- Pre-wet the phosphocellulose filter plate by adding ~50μlof
2% (v/v) phosphoric acid and filter by applying vacuum using
the vacuum manifold (seeNote 42).
- Transfer the quenched AMPK reaction mixture to the filter
plate and filter by applying vacuum. Wash the wells six times
with 75μl of wash buffer.
- Allow filter plate to dry. Seal the bottom of the plate using a
plate seal and add 25μl of scintillation fluid.
- Seal the top of the plate and count the radioactivity on an
instrument such as a PerkinElmer MicroBeta Trilux. The signal
in each well (CPM) is directly proportional to the amount of
phosphorylated peptide produced (seeNote 43).
- Plot the signal from each well as a function of time to generate a
reaction progress curve. The slope of the initial linear portion
of this time course is used to determine the rate of each
reaction.
- Plot the reaction rates at different concentration of peptide as a
function of the concentration of peptide. Fit the data in this
hyperbolic plot to the Michaelis-Menten equation using
GraphPad Prism or a similar software to determine theVmax
andKm. Compare these kinetic parameters with and without
compound treatment to determine the direct effects of the
allosteric activators onVmaxandKm[9, 10](seeNote 44).
Biophysical Studies to Evaluate Protein-Ligand Interactions 45