AMPK Methods and Protocols

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 4
when added to the reaction, the

final (ATP) in the reaction will be 10
the ATPKmvalue. 4 


SAMS peptide substrate mixture—Prepare SAMS peptide solu-

tion by diluting SAMS peptide in 1
kinase buffer to a con-

centration equal to 40
the 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.

2. 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.


3. Add 10μl of the ATP mixture.


4. 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.


5. 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).


6. 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.


7. Allow filter plate to dry. Seal the bottom of the plate using a
   plate seal and add 25μl of scintillation fluid.


8. 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).


9. 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.


10. 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