Figure 15. Radioactive flux assays of... | Rockefeller University Press

Figure 15.

$Radioactive flux assays of wild-type and mutant AdiC. (A and B) Time courses of radioactive Arg+ uptake from the bathing solution into the lipid vesicles embedded with the wild-type (A) and the mutant (B) AdiC molecules. The initial concentration of nonradioactive Arg+ inside the vesicles was 50 mM in all cases. At the time t = 0, radioactive Arg+ at a concentration 0.5 mM or 50 μM in A and 50 μM in B were added to the bathing solution to initiate the transport process. The graph in B is plotted for a >10-fold longer period in the inset. Data points are presented as fractional uptakes (mean ± SEM), defined as radioactive Arg+ inside the vesicles as a fraction of the total radioactive Arg+ in the sample. The number of assays were 3–6, except 2 for the leftmost data point for the 0.5 mM condition in A. The black dashed lines indicated the theoretically maximum fractional uptake expected for a 1:1 exchanger under the corresponding conditions. The smooth curve (green) overlaid on the data in B is calculated with the model, as described in Results and in Supplemental material. Given the sidedness of AdiC is random in the vesicle, the average of simulations with two models in Figs. S6 and S7 is plotted, labeled as simulation of a uniporter model. (C and D) Uptake of radioactive Arg+ with different initial Arg+ concentrations in the bathing solution while the initial concentration of nonradioactive Arg+ in the vesicles was always 50 mM. The fractional uptakes (mean ± SEM; n = 3) via the wild-type (C) or the mutant (D) AdiC molecules at t = 4 h are represented by red dots, plotted against the concentration of Arg+ in the bathing solution (upper x axis) or against the ratio of initial Arg+ concentrations in the bathing solution and in the vesicles (lower x axis). The blue dots are the theoretical maximum uptake predicted for a 1:1 exchanger under the corresponding substrate conditions. The green dots in D represent the fractional uptakes (mean ± σ) simulated by means of a Monte Carlo method, using our model with an apo path for conformational transitions, which is labeled as simulation of a uniporter model, as described above. An equal ratio of the two configurations of the model was used in all simulations to mimic the random insertions of AdiC in the two opposite membrane orientations in the flux assays.$

Radioactive flux assays of wild-type and mutant AdiC. (A and B) Time courses of radioactive Arg⁺ uptake from the bathing solution into the lipid vesicles embedded with the wild-type (A) and the mutant (B) AdiC molecules. The initial concentration of nonradioactive Arg⁺ inside the vesicles was 50 mM in all cases. At the time t = 0, radioactive Arg⁺ at a concentration 0.5 mM or 50 μM in A and 50 μM in B were added to the bathing solution to initiate the transport process. The graph in B is plotted for a >10-fold longer period in the inset. Data points are presented as fractional uptakes (mean ± SEM), defined as radioactive Arg⁺ inside the vesicles as a fraction of the total radioactive Arg⁺ in the sample. The number of assays were 3–6, except 2 for the leftmost data point for the 0.5 mM condition in A. The black dashed lines indicated the theoretically maximum fractional uptake expected for a 1:1 exchanger under the corresponding conditions. The smooth curve (green) overlaid on the data in B is calculated with the model, as described in Results and in Supplemental material. Given the sidedness of AdiC is random in the vesicle, the average of simulations with two models in Figs. S6 and S7 is plotted, labeled as simulation of a uniporter model. (C and D) Uptake of radioactive Arg⁺ with different initial Arg⁺ concentrations in the bathing solution while the initial concentration of nonradioactive Arg⁺ in the vesicles was always 50 mM. The fractional uptakes (mean ± SEM; n = 3) via the wild-type (C) or the mutant (D) AdiC molecules at t = 4 h are represented by red dots, plotted against the concentration of Arg⁺ in the bathing solution (upper x axis) or against the ratio of initial Arg⁺ concentrations in the bathing solution and in the vesicles (lower x axis). The blue dots are the theoretical maximum uptake predicted for a 1:1 exchanger under the corresponding substrate conditions. The green dots in D represent the fractional uptakes (mean ± σ) simulated by means of a Monte Carlo method, using our model with an apo path for conformational transitions, which is labeled as simulation of a uniporter model, as described above. An equal ratio of the two configurations of the model was used in all simulations to mimic the random insertions of AdiC in the two opposite membrane orientations in the flux assays.