Table 1. Allosteric activation of AMPK complexes by 991.
AMPK complexes expressed in β1β2 dKO cells were isolated by immunoprecipitation using FLAG affinity resin. After extensive washing, proteins were eluted using the FLAG peptide and AMPK activity was measured using the SAMS peptide assay in the presence of varying concentrations of 991. The data were fitted to the equation: v = b + {[(s × b) − b] × [991]}/(A0.5 + [991]), where s is the relative stimulation, b is the basal activity and A0.5 is the concentration of 991 giving half-maximal stimulation, using the GraphPad Prism software. In all cases, results are from at least three independent experiments and are plotted as the means ± SEM.
| Complex | A0.5 991 (nM) | Activation (fold) |
|---|---|---|
| α2β2γ1 | 147 ± 28 | 5.7 ± 0.3 |
| α2β2γ2 | 97 ± 18 | 5.0 ± 0.2 |
| α2β2(S108A)γ2 | 170 ± 35 | 5.4 ± 0.3 |
| α2β1γ2 | 12.7 ± 4 | 7.7 ± 0.4 |
| α2β1(S108A)γ2 | 462 ± 142 | 5.2 ± 0.5 |