Table 1. Allosteric activation of AMPK complexes by 991 and A-769662.
| Complex |
Activation (fold) |
A0.5
(μM) |
|||
|---|---|---|---|---|---|
| 991 | A-769662 | AMP | 991 | A-769662 | |
| α1β1γ1 | 4.8 (0.1) | 2.0 (0.1) | 1.5 (0.1) | 0.03 (0.01) | 0.59 (0.07) |
| α1β2γ1 | 2.4 (0.1) | No activation | 1.4 (0.1) | 1.1 (0.3) | ND |
| α2β1γ1 | 12.2 (0.6) | 14.3 (0.4) | 2.6 (0.1) | 0.09 (0.02) | 0.39 (0.03) |
| α2β2γ1 | 5.4 (0.4) | No activation | 3.0 (0.2) | 0.51 (0.19) | ND |
| ΔCBM | No activation | No activation | 2.2 (0.1) | ND | ND |
| α2(K29/K31)β1γ1 | 4.0 (0.6) | No activation | 1.5 (0.1) | 2.3 (0.8) | ND |
| α2β1(S108A)γ1 | 11.6 (2.0) | No activation | 2.1 (0.1) | 3.6 (1.2) | ND |
| α1β1(R83A)γ1 | No activation | No activation | 1.4 (0.1) | ND | ND |
| α2β1(L166E)γ1 | 2.0 (0.1) | No activation | 1.8 (0.1) | 0.12 (0.04) | ND |
AMPK activity was measured using the SAMS peptide assay. Allosteric activation by 0.1 mM AMP is also shown. In all cases, results shown are the mean (±s.e.m.) determined from at least three independent experiments. In some cases, no activation by compound was detectable up to concentrations of 10 μM, and in these cases the A0.5 was not determined (ND). Concentrations of 991 or A-769662 above 10 μM inhibited AMPK possibly through a deleterious effect on the enzyme. The specific activities of the various AMPK complexes (following phosphorylation by CaMKKβ) were similar, and, in the absence of 991, A-769662 or AMP was within a range varying between 300 and 500 nmol−1min−1mg−1. The activity of the α2β1(R83A)γ1 complex was too low to measure accurately and so is not included here. As a result the α1β1(R83A)γ1 activity data are included. ΔCBM, AMPK complex (α1β1(185-270)γ1) lacking the carbohydrate-binding module (CBM).