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Figure 3. L-leucine uptake by endogenous Xenopus laevis oocyte transporters.Three minute uptakes by non-injected (NI) oocytes of 10–1000 μM L-leucine (Leu) in uptake buffers containing (+Na+) and without sodium (Na+free) were tested using radiolabeled amino acid (AA) tracers. In all panels uptake data (pmol/3 min per oocyte) were normalized to uptake in 1 mM Leu, +Na+ uptake buffer for each batch of oocytes and experimental day. Panel (A) shows normalized Leu uptake rate data (expt) vs. the simultaneous fit (fit) for endogenous Leu uptake rates. Panel (B) shows model calculations (model) for contributions to uptake of 10–1000 μM Leu by various endogenous Xenopus laevis oocyte (xAAT) species. Model output was calculated based on the calculated xAAT Vmax,i and the reported Km for each xAAT ortholog (Table 1). The 95% confidence limits for transporter activities are shown (dotted lines) bracketing the model predictions. Panels (C,D) show the calculated Leu uptake rates in +Na+ and Na+ free uptake buffers containing excess competitive inhibitors. Panel (C) shows the concentration dependence (0–1000 μM Leu) of normalized cumulative uptake data by NI oocytes in Na+free uptake buffers containing 10 mM each of the following competitors: L-alanine (Ala) (Na+free+Ala), Ala and L-tryptophan (Trp) (Na+free+Ala+Trp), L-valine (Val) (Na+free+Val) vs. calculated cumulative endogenous uptake results for the respective uptake buffers. For uptakes in Na+free+Ala, and Na+free+Ala+Trp uptake buffers, the calculated values were virtually indistinguishable from the global fit for the Na+free data, therefore a single line was used for graphing all three data sets. Panel (D) shows the concentration dependence (0–1000 μM) of total Leu uptake rates in +Na+ uptake buffers containing 10 mM each of the following competitors: Ala, Arg (+Na++Ala+Arg), 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH) (+Na++BCH), and BCH and Ala (+Na++BCH+Ala) (Fig. 2). n = 6–8 ooyctes each experiment, for 3 independent experiments. The experimental data are shown as the mean ± SEM for the measured uptake rates and the calculated cumulative endogenous uptake rates were based on the previously calculated Vmax and reported Km values for the kinetic components exhibited by each AAT species (Table 1). Image published in: Taslimifar M et al. (2017) Copyright © 2017, The Author(s). This image is reproduced with permission of the journal and the copyright holder. This is an open-access article distributed under the terms of the Creative Commons Attribution license Larger Image Printer Friendly View

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