Results from TSR analysis are valid across a broad range of competing-substrate concentration ratios The Transport Specificity Ratio (TSR) is calculated using results from a dual-label competitive uptake assay in which structurally distinct, labelled substrates are allowed to compete for transport at the same active site. Panel A: Mixtures of 10 μM [3H]NA (0.6 μCi/ml) and 10 μM [14C]GABA (0.2 μCi/mL) were prepared such that [NA] + [GABA] = 10 μM. E. coli strains SK105 (GabP-positive) and SK45 (GabP-negative) were exposed in parallel experiments for 10 seconds at 30°C to substrate mixtures containing the indicated concentrations of [3H]NA. The GabP-dependent (SK105 minus SK45) uptake of either [3H]NA (■) or [14C]GABA (▲) may be read from the left-side ordinate. The calculated TSR (Equation. 6) may be read from the right-side ordinate (○). Panel B: The substrate concentrations were varied in constant proportion such that the GABA concentration (ranging from 1.8–31.5 μM) was always 42.9 percent of the NA concentration (ranging from 4.2–73.4 μM). The radiochemical concentrations for [3H]NA and [14C]GABA were 0.23 μCi/ml and 0.03 μCi/ml, respectively. The indicated concentration ranges produce about 50 percent combined active site occupancy (bound GABA plus NA) – since the affinities for GABA and NA are 40 μM and 200 μM, respectively .