A fluorescent galactoside, 2-(N-dansyl)-aminoethyl beta-D-thiogalactoside (dansyl-galactoside), competitively inhibits lactose transport by membrane vesicles of Escherichia coli, but is not actively transported. An increase in dansyl-galactoside fluorescence is observed upon addition of D-lactate. The fluorescence increase is not observed in membrane vesicles lacking the beta-galactoside transport system, and is blocked or rapidly reversed by addition of beta-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 dansyl-galactoside molecules are excited by energy transfer from the membrane proteins. Titration of vesicles with dansyl-galactoside in the presence of D-lactate demonstrates that the beta-galactoside carrier protein represents about 3.3% of the total membrane protein. The data indicate that D-lactate oxidation leads to binding of the fluorescent galactoside to the beta-galactoside carrier protein in such a manner that the dansyl group is transferred to a hydrophobic environment within the membrane.

Binding Sites↗

PubMed1973

Anaerobic transport in Escherichia coli membrane vesicles.

Anaerobic beta-galactoside transport in whole cells and membrane vesicles from E. coli ML 308-225 is coupled to the oxidation of alpha-glycerol-P or D-lactate with fumarate as an electron acceptor. Alternatively, anaerobic beta-galactoside transport may be coupled to the oxidation of formate utilizing nitrate as electron acceptor. Both anaerobic electron-transfer systems are induced by growth of the organisms under appropriate conditions. Components of both systems are loosely bound to the membrane, necessitating the use of a modified procedure for vesicle preparation in order to demonstrate anaerobic transport in vitro. Addition of ATP or an ATP-generating system to vesicles prepared from anaerobically-grown cells or inclusion of ATP or the ATP-generating system during preparation of vesicles does not stimulate transport. The results support the conclusion that active transport under anaerobic conditions is coupled primarily to electron flow.

Adenosine Triphosphate↗

PubMed1973

Reconstitution of D-lactate-dependent transport in membrane vesicles from a D-lactate dehydrogenase mutant of Escherichia coli.

Membrane-bound, flavin-linked D-lactate dehydrogenase in membrane vesicles of E. coli ML 308-225 is solubilized by extraction with guanidine HCl. When membrane vesicles prepared from a D-lactate dehydrogenase mutant are treated with this extract, they regain the capacity to catalyze D-lactate oxidation and D-lactate-dependent transport. Similar effects are obtained with wild-type membrane vesicles in which D-lactate oxidation and D-lactate-dependent transport have been inactivated by 2-hydroxy-3-butynoate. Although treatment of wild-type vesicles with the extract results in an increased capacity to catalyze D-lactate oxidation, no effect on transport is observed. Reconstituted transport activity is a saturable function of the amount of guanidine extract added. Moreover, the quantity of extract required to achieve maximum initial rates of transport varies with each transport system. On the other hand, reconstituted D-lactate oxidation increases linearly over a broader range of extract concentrations.Oxamate, a competitive inhibitor of D-lactate dehydrogenase, and p-chloromercuribenzenesulfonate block both the initial rate of transport and the steady-state level of accumulation in reconstituted vesicles. Furthermore, these reagents induce efflux of transport substrates from preloaded, reconstituted vesicles. The same reagents inhibit the initial rate of uptake but not the steady-state level of accumulation in ML 308-225 vesicles, and do not induce efflux. These results suggest that, although reconstituted vesicles catalyze D-lactate oxidation and D-lactate-dependent transport, the system has not been reconstituted to its native state.

Amino Acids↗

PubMed1973