Energy-dependent Binding of Dansylgalactosides to the P-Galactoside Carrier Protein*

Fluorescent P-galactosides (l-(N-dansyl)amino-P-D-galactopyranoside (DGo), 2’-(N-dansyl)aminoethyl-/&D-thiogalactopyranoside (DG*), 2’-(N-dansyl)aminoethyl-&D-galactopyranoside (oxy-DG2), and 6’-(N-dansyl)aminohexyl-b-othiogalactopyranoside (DGs)) competitively, inhibit lactose transport by membrane vesicles from Escherichia coli ML 308-225, but are not actively transported. An increase in the fluorescence of these dansylgalactosides is observed upon addition of D-lactate, imposition of a membrane diffusion potential (positive outside), or dilution-induced, carriermediated lactose ef%x. The increase is not observed with 2’-(N-dansyl)aminoethyl-p-o-thioglucopyranoside nor with membrane vesicles lacking the /3-galactoside transport system. Moreover, the o-lactate-induced fluorescence increase is blocked or rapidly reversed by addition of /3-galactosides, sulfhydryl reagents, inhibitors of D-lactate oxidation, or uncoupling agents. The fluorescence increase exhibits an emission maximum at 500 nm and excitation maxima at 345 nm and at 292 nm. The latter excitation maximum is absent unless D-lactate is added, indicating that the bound dansylgalactoside molecules are excited by energy transfer from the membrane proteins. Titration of vesicles with dansylgalactosides in the presence of D-lactate demonstrates that the lac carrier protein constitutes 3 to 4% of the total membrane protein, and that the affinity of the carrier for substrate is directly related to the length of the alkyl chain between the galactosidic and the dansyl moieties of the dansylgalactosides. In addition, there is excellent agreement between the affinity constants of the various dansylgalactosides as determined by fluorimetric titration and their apparent KS for lactose transport (K,s and/or apparent Kis are approximately 550, 30, 40, and 5 ~IH for DGo, DG2, oxy-DGt, and DGs, respectively). Polarization of fluorescence measurements with DG2 and DGs demonstrate a dramatic increase in polarization on addition of D-lactate which is reversed by addition of lactose or anaerobiosis. These findings provide strong evidence for