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Nucleotide binding to wild-type AMPK. A and B, competition of 0.5 μm deac-ADP bound to α1β2γ1 AMPK by unlabeled Mg2+-AMP (A) and Mg2+-ATP (B). The dashed line indicates the fluorescence signal of unbound deac-ADP. Because fluorescence competition failed to unambiguously determine the IC50 value for the high affinity AMP-binding site, we independently determined its affinity by ITC (C).
