Fig 6.

Transport kinetics and cosubstrate-binding affinity. (A) K_m and V_max for melibiose transport. Cell preparation and transport assay were performed as described in Materials and Methods. Fitted initial rates of melibiose transport at a given melibiose concentration between 0.05 and 2.5 mM (specific activity, 3.2 to 10 mCi/mmol) were corrected by the rates obtained from nontransformed DW2 cells. Means (± standard errors [SE]) from 2 to 5 tests were plotted as a function of melibiose concentration. (B) Apparent affinity for Na⁺ binding. Determination of K_0.5^Na+ for the D²G FRET was carried out with RSO vesicles (0.5 mg/ml; 100 mM KP_i, pH 7.5) containing the WT or a MelB_St mutant at excitation and emission wavelengths of 290 and 500 nm, respectively. The mean values of ΔI/I₀ (%) with SE and/or the values from 2 to 3 tests were plotted as a function of Na⁺ concentration. (C) Apparent affinity for melibiose binding. Determination of IC₅₀ for melibiose displacement of bound D²G was performed with RSO vesicles containing 10 μM D²G and 200 mM NaCl. The mean values of the melibiose-induced change in intensity (ΔF) with SE and/or data from 2 to 3 tests were plotted as a function of melibiose concentration. The values for K_m, V_max, K_0.5^Na+, and IC₅₀ were determined by fitting a hyperbolic function to the data (OriginPro 8.6).