Figure 5
From: Identification of a High-Affinity Pyruvate Receptor in Escherichia coli
Analysis of the interaction of BtsS with selected ligands by DRaCALA. (A) A mixture of membrane vesicles (MV) or right-side-out vesicles (RSO) enriched with the corresponding proteins (indicated by graphical representations) and radiolabeled 14C pyruvate (5 µM) is dropped onto a nitrocellulose membrane, and ligand migration via capillary action is analyzed. (B) Competition assays. Binding of radiolabeled pyruvate (5 µM) to BtsS in MVs was analyzed in the presence of various unlabeled competitors (each 50 mM). NC, no competitor. (C) Relative efficiency of competition by various carboxylic acids. Binding of radiolabeled pyruvate (5 µM) to BtsS in MVs was analyzed in the presence of various carboxylic acids (each 50 mM). The efficiency of competition by cold pyruvate was set to 1.00, and the effect of the indicated compounds was calculated accordingly. (D) Determination of the dissociation constant (K d ) for pyruvate to BtsS using DRaCALA. For each reaction radiolabeled pyruvate was used at 5 µM. Normalized FB values [FB = (FB(NC) − FB(pyr))/FB(NC), see Methods for details] were plotted as function of the pyruvate concentration. The best-fit line was determined by nonlinear regression using the equation y = Bmax * x/(K d + x).
