Figure 9.

NADPH binds adenine nucleotide-binding site(s) in the γ-subunit. A, NADPH emission spectra in the absence and presence of MBP and MBP-tagged phosphorylated α1β2γ1 AMPK. The spectrum of AMPK in the absence is shown as negative control. B, estimation of NADPH and NADH binding constants. 5 μm NADPH or NADH were incubated with increasing concentrations of MBP-AMPK and MBP-AMPK concentrations were plotted against fluorescence intensity at the emission maxima. C and D, AMP and ATP compete the NADPH binding signal. Emission spectra of 5 μm NADPH, 10 μm AMPK were recorded in the absence and presence of increasing concentrations of AMP (C) and ATP (D).