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. 2004 Aug 10;101(33):12148–12152. doi: 10.1073/pnas.0404936101

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Copyright © 2004, The National Academy of Sciences

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Fig. 3. — Lactose protection against [¹⁴C]NEM labeling of single-Cys-148 LacY. (A and B) Ice-cold RSO or ISO vesicles containing single-Cys-148 mutant at 10 mg of protein per ml were added to a solution containing [¹⁴C]NEM (40 mCi/mmol, 0.5 mM final concentration) and given concentrations of lactose and incubated on ice for 5 min. Reactions were quenched with 10 mM dithiothreitol, and biotinylated LacY was purified and analyzed as described for Fig. 2. Labeling in the presence of a given concentration of lactose is expressed as percent labeling observed in the absence of ligand. (C and D) Effect of on the lactose protection against Cys-148 alkylation by NEM. Ice-cold RSO or ISO membrane vesicles were added with 20 mM lithium d-lactate under oxygen or 10 mM Mg(II)ATP, respectively, and incubated on ice for 5 min before addition to a solution containing [¹⁴C]NEM and given concentrations of lactose as described above (final protein concentration, 10 mg/ml). K_D values were calculated as described in Materials and Methods.

Inline graphic — Lactose protection against [¹⁴C]NEM labeling of single-Cys-148 LacY. (A and B) Ice-cold RSO or ISO vesicles containing single-Cys-148 mutant at 10 mg of protein per ml were added to a solution containing [¹⁴C]NEM (40 mCi/mmol, 0.5 mM final concentration) and given concentrations of lactose and incubated on ice for 5 min. Reactions were quenched with 10 mM dithiothreitol, and biotinylated LacY was purified and analyzed as described for Fig. 2. Labeling in the presence of a given concentration of lactose is expressed as percent labeling observed in the absence of ligand. (C and D) Effect of on the lactose protection against Cys-148 alkylation by NEM. Ice-cold RSO or ISO membrane vesicles were added with 20 mM lithium d-lactate under oxygen or 10 mM Mg(II)ATP, respectively, and incubated on ice for 5 min before addition to a solution containing [¹⁴C]NEM and given concentrations of lactose as described above (final protein concentration, 10 mg/ml). K_D values were calculated as described in Materials and Methods.