Figure 7. Inositol competes with AMP in binding to AMPKγ and represses AMPK kinase activity.

(A) AMPK kinase activity upon Ins treatment was determined by immunoblotting with pMff. (B) AMPK kinase activity was determined by ADP-Glo^™ kinase assay. (C) AMPK kinase activity was determined by immunoblotting with pMff. (D) The effects of Ins on AMPK activity using SAMS peptide were determined by fitting Michaelis-Menten equation. K_i for inositol was calculated by using Michaelis-Menten equation. (E) AMPK kinase activity with fixed amount of ATP upon Ins or ATP treatment was determined by immunoblotting with pMff. (F) AMPK kinase activity with fixed amount of ATP upon adding Ins with or without ATP was determined by immunoblotting with pMff. (G) AMPK kinase activity upon adding Ins, AMP or ATP was determined by immunoblotting with pMff. (H) Bar graphs represent each concentration of AMP with or without Ins in AMPK enzymatic kinetic curve. (I and M) Immunoblotting of biotin pull-down assay incubated with recombinant fusion proteins. (J) Saturation binding of [³H]inositol with recombinant GST-AMPKγ1 proteins. Dissociation constant (K_d) value of 98.2 pmol ± 2.8 pmol was determined for binding affinity between Ins and AMPKγ1. (K) Coomassie blue staining of the indicated recombinant proteins. GST-Bateman domain 1 (BD1) and Bateman domain 2 (BD2). (L) The binding between Biotin-Ins and indicated recombinant proteins was determined by immunoblotting. (N) The competitive binding assay was subjected to immunoblotting with specific AMPKγ antibody. (O) Competition binding of [³H]inositol (3.75 μM) and AMP with recombinant GST-AMPKγ1 protein (100 ng). IC₅₀ = 1.9 μM ± 0.5 μM. (P) Competition binding of [³H]inositol (3.75 μM) and ATP with recombinant GST-AMPKγ1 protein. (Q) The competitive binding assay between AMP and Ins was determined upon incubation of 3.33 μM of tritium-labeled AMP (³H-AMP) and indicated recombinant proteins.