Figure 3.

Positional isotope exchange with commercial human P-gp supersomes. Total proteins (28.2 μg/mL) were incubated with 2 mM γ-¹⁸O₄-ATP, 50 mM Tris·HCl, 15 mM NH₄Cl, 5 mM MgSO₄, 2.5 mM EGTA, 0.02 wt % NaN₃, 2 mM TCEP, pH 7.4 in the presence of 50 μM nicardipine. For the samples with inhibitors (gray spectra top middle panel), conditions were as described in Materials and Methods. ³¹P NMR spectra were recorded at the times indicated. In the left-hand inset, the ³¹P_γ signals of ATP bearing four or three ¹⁸O are presented in blue and red, respectively. The ³¹P_β signals from ADP clearly demonstrate that ADP is formed concomitant with the changes in isotopic composition of the ³¹P_γ signal (red vs blue). In the right-hand inset, the ³¹P_β signals originating from ATP with ¹⁸O at the bridging position (¹⁸O_B) or at the nonbridging position (¹⁸O_NB) are shown in blue and red, respectively.