Comparison of the observed and calculated rates of the AdiC-mediated uptake of Arg ⁺ into lipid vesicles in the outside-out configuration. (A and B) Observed uptake rates of Arg⁺ (closed black circles; mean ± SEM) (Tsai et al., 2012) and the predicted rates by the model (open pink circles; mean ± SD), plotted against the Arg⁺ concentration. In panel A, C₂ being assigned as E_o and C₃ as I_o (Fig. S6), whereas in panel B, C₂ as I_o and C₃ as E_o (Fig. S7). The solid curves superimposed on symbols representing the calculated values correspond to the fits of the Michaelis–Menten equation. In the presence of near saturating concentrations of a given type of substrate on both sides of the membrane, the observed maximal net-flux rates (k_max) are expected to be comparable in both orientations of AdiC. Indeed, under the three conditions that differ in substrate type or AdiC orientation, k_max values are comparable (Fig. 12). However, observed k_max for Arg⁺ in the outside-out orientation shown here is much lower, which reflects an underestimate of the maximal transporting rate or an overestimate of the effective number of AdiC molecules or both. Our calculations, as expected, yield comparable k_max values for all four conditions. The divergence between observed and calculated k_max values in the outside-out configuration is illustrated graphically by overlaying the observed data and the thin pink curve that represents a Michaelis–Menten equation fit to the calculated data in A or B after scaling down.