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A fluorescent galactoside, 2-(N-dansyl)-aminoetliyl O-i-tlhiogalactosidc (dansyl-galactoside), competitively inhibits lactose transport by membrane vesicles of Escherichia coli, but is not actively transported. An increase iii dlansl-galactoside fluorescence is observed upon addition of D-laetate. Ihc The sampale chamber wvas maintained at 23°C wxith a circulating xwater bath. Spectra were not corrected for variatioiis ii1 either ilatenisitx of the light source or reslponise of the photomiultiphlier with xaxvelength. Additiosis to the cuvette were made wsith Hamilton microsyringes. Samples were mixesi within 2-3 see with a small lplastic stick.Lactose Transport by membrane vesicles wxaxs assayed at a final concentration of 0.4 miM (3,4,21).N-Ethy/onaleimide (NE.l1) Protection Experimnents. Membraise vesicles were incubated for 5 miss with 1.0 inM A'-ethvlmaleimide iin the presenice of various cosicentrationIs of dansyl-galactoside. After the reactioiis were stopped by addition of dithiothreitol (10 mM\1 final concentration), the vesicles 2722

High specific activity 6'-N43Hjdansyl)aminohexyl 1-thio-fl-D-galactopyranoside Recent studies with (N-dansyl)aminoalkyl 13-D-galactopyranosides (1-4) and (2-nitro-4-azidophenyl)13-D-galactopyranosides (5, 6) indicate that the lac carrier protein in membrane vesicles isolated from Escherichia coli does not bind ligand significantly in the absence of a membrane potential (interior negative). As a result of D-lactate or reduced phenazine methosulfate oxidation, or artificially induced ion gradients, an electrical potential is generated across the vesicle membrane (7-10), and changes in dansylgalactoside fluorescence and azidophenylgalactoside-dependent photoinactivation are observed. Alternatively, dilution-induced, carriermediated lactose efflux also causes changes in dansylgalactoside fluorescence in a manner which is apparently independent of the membrane potential (2). Based on these observations, it has been postulated (2) that the membrane potential causes the lac carrier protein to become accessible to the external medium, to increase its affinity for ligand, or both; and it has been suggested that the lac carrier protein or a part of it may be negatively charged.The strength of these conclusions rests heavily on the contention that the fluorescence changes observed with the dansylgalactosides are due specifically to binding and not to a subsequent translocational event, and this aspect of the problem has been approached in several ways. It has been demAbbreviations: Dns2-Gal, 2'-(N-dansyl)aminoethyl 1-thio-fl-Dgalactopyranoside; Dns6-Gal, 6'-(N-dansyl)aminohexyl 1-thio-f-Dgalactopyranoside (in previous papers of this series, the abbreviations DG2 and DG6 were used); CCCP, carbonylcyanide m-chlorophenylhydrazone; p-CMBS, p-chloromercuribenzenesulfonate; TDG, (3--galactosyl 1-thio-,B-galactopyranoside. * This is paper XXXI in the series "Mechanisms of Active Transport in Isolated Bacterial Membrane Vesicles." Paper XXX is given in ref. 4. onstrated that there is a marked increase in fluorescence anisotropy of 2'-(N-dansyl)aminoethyl 1-thio-13-D-galactopyranoside (Dns2-Gal) on "energization" of membrane vesicles containing the lac carrier protein (2, 4), and that the lifetime of its excited-state is increased when the molecule is bound (4). Concurrently, the rotational diffusion of Dns2-Gal is dramatically decreased (4). In addition, it has been demonstrated by various means that the dansylgalactosides are not transported to any extent (1-3), and finally that p-chloromercuribenzenesulfonate (p-CMBS) causes rapid reversal of D-lactate-induced dansylgalactoside fluorescence although it does not cause efflux of lactose from the intravesicular pool (3). The latter finding indicates that the changes in dansylgalactoside fluorescence observed on energization cannot be attributed to binding followed by translocation into the hydrophobic milieu of the membrane. As discussed previously (9), these conclusions represent a significant departure from previous notions regarding the mechanism of active transport...

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