Figure 3. Allosteric activation of AMPK-γ isoforms at different concentrations of ATP.

Cells expressing FLAG–γ1,–γ2 or–γ3 were treated with 10 mM phenformin for 60 min to activate AMPK and the different complexes were immunoprecipitated using anti-FLAG antibody. Kinase assays were conducted at the indicated concentrations of AMP, but with ATP at 200 μM (A), 5 mM (B) or 20 μM (C). MgCl₂ was also present at a concentration 4.8 mM higher than ATP; this protocol results in the proportions present as free ATP and as the MgATP²⁻ complex remaining approximately constant [55]. Results (means ± S.E.M., n=3) are expressed relative to the basal activity without AMP and were fitted to the equation: Y=1+{[(activation–1) × X]/(EC₅₀+X)}–{[(activation) × X]/(IC₅₀+X)}, where Y is the activity, X is the concentration of AMP, activation is the predicted maximal activation for the activating phase of the curve, EC₅₀ is the concentration of AMP causing half-maximal activation, and IC₅₀ is the concentration of AMP causing half-maximal inhibition. The continuous lines are the curves generated by this equation using the values for activation, EC₅₀ and IC₅₀ quoted in the text.