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. 2014 Feb 12;53(9):1536–1543. doi: 10.1021/bi401716z

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Transport kinetics of WT LacY and selected N272 mutants. E. coli T184 cells expressing WY LacY (●), N272D (○), N272V (△), or N272Q (□) in 0.1 M KP_i (pH 7.5) and 10 mM MgSO₄ at an OD₄₂₀ of 10 (50 μL) were incubated with [1-¹⁴C]lactose at a given concentration at room temperature for 20 s as described in Materials and Methods. The samples were rapidly diluted with 3 mL of stop buffer and vacuum filtered. The filters were washed once with 3 mL of stop buffer and assayed for radioactivity by liquid scintillation spectrometry. K_m and V_max values were determined with GraFit version 6 (Erithacus Software) using the Michaelis–Menten equation (Table 1):

graphic file with name bi-2013-01716z_m002.jpg — Transport kinetics of WT LacY and selected N272 mutants. E. coli T184 cells expressing WY LacY (●), N272D (○), N272V (△), or N272Q (□) in 0.1 M KP_i (pH 7.5) and 10 mM MgSO₄ at an OD₄₂₀ of 10 (50 μL) were incubated with [1-¹⁴C]lactose at a given concentration at room temperature for 20 s as described in Materials and Methods. The samples were rapidly diluted with 3 mL of stop buffer and vacuum filtered. The filters were washed once with 3 mL of stop buffer and assayed for radioactivity by liquid scintillation spectrometry. K_m and V_max values were determined with GraFit version 6 (Erithacus Software) using the Michaelis–Menten equation (Table 1):