Figure 1.

Binding of IIA^glc to lactose permease in the presence of various sugars. Membrane vesicles were prepared from strains T184/pT7–5 (control) and T184/pT7–5/cassette lacY (LacY) harvested 4 h after induction with 1 mM isopropyl 1-thio-β-d-galactopyranoside as described in Materials and Methods. Membrane vesicles (0.5 mg protein) were mixed with 1.2 μg of [³H]IIA^glc (1080 cpm/μg of protein) in 50 mM Na⋅phosphate buffer, pH 6.3, containing 2 mM DTT and 2 mM MgCl₂ in total volume of 100 μl in the presence or absence of 2 mM sugars as indicated. After incubation at room temperature for 5 min, the membranes were separated in an Airfuge and the bound and free IIA^glc were determined as described in Materials and Methods. The inset shows the TDG-dependent binding between IIA^glc and membrane vesicles enriched in lac permease as a function of membrane protein concentration. TDG-dependent binding was determined by subtracting the ³H counts in the membrane pellet of the incubation mixture without added sugar from the ³H counts of membrane-bound [³H]IIA^glc in the presence of TDG. Binding assays were done more than twice and SD was <5%.