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A hybrid between the maltose-binding protein (MalE) of Escherichia coli and the gene 5 protein (G5P) of phage M13 was constructed at the genetic level. MalE is a monomeric and periplasmic protein while G5P is dimeric and cytoplasmic. The hybrid (MalE-G5P) was synthesized in large amounts from a multicopy plasmid and efficiently exported into the periplasmic space of E. coli. The export was dependent on the integrity of the signal peptide. MalE-G5P was purified from a periplasmic extract by affinity chromatography on cross-linked amylose, with a yield larger than 50000 molecules/E. coli cell. The hybrid specifically bound denatured but not double-stranded DNA cellulose, as native G5P. Sedimentation velocity and gel-filtration experiments showed that MalE-G5P exists as a dimer. Thus, it was possible to efficiently translocate through the membrane a normally cytoplasmic and dimeric protein, by fusion to MalE. Moreover, the passenger protein kept its activity, specificity and quaternary structure in the purified hybrid. MalE-G5P will enable the study of mutant G5P that no longer binds single-stranded DNA and therefore cannot be purified by DNA-cellulose chromatography. [ABSTRACT FROM AUTHOR]

1. Accumulation of substrate via the maltose transport system of Escherichia coli is incompletely inhibited by uncouplers of oxidative phosphorylation. The extent of this inhibition is reduced upon introduction of a mutation in the Mg2+ Ca2+-dependent ATPase. This suggests that lack of importance of the proton motive force for accumulation of substrate. 2. Starved cells of the ATPase mutant show a reduced rate of maltose uptake. This low rate was strongly stimulated by the glycolytic carbon source D-fructose but not by oxidisable energy sources such as D-lactate or ascorbate plus phenazine methosulphate. These findings are also in agreement with the lack of importance of the proton motive force in maltose accumulation. 3. Incubation of wild-type as well as ATPase-less cells with high concentrations of arsenate, that is known to deplete the intracellular level of ATP in E. coli, did not significantly inhibit the uptake of maltose. This suggests that the level of intracellular ATP is not directly the energy source for maltose uptake. 4. The exit of maltose, in both the wild-type and the ATPase-less strains, is inhibited by uncouplers of oxidative phosphorylation. Azide, dinitrophenol, and carbonyl cyanide chlorophenyl-hydrazone inhibited the downhill exit of maltose and also the exchange of maltose; arsenate did not. This suggests a requirement for the proton motive force in transport of maltose in the outward direction, and contrasts from the properties of the entry reaction. These results are consistent with the view that entry and exit of maltose are mediated via different carriers and energized by different modes. [ABSTRACT FROM AUTHOR]

The binding protein-dependent transport of galactose and maltose occurs at a reduced but significant rate in Escfwrichia cofi cells which have undergone a mild toluenization. Dihydrolipoate and 3-acetyl-NAD produce a severalfold stimulation of these transports in the toluenized cells. In parallel to the stimulation of galactose and maltose transport by dihydrolipoate and 3-acetyl-NAD, there is a stimulation by galactose and maltose of lipoamide dehydrogenase activities which seem to be related to the binding-protein-dependent transport of these sugars. The lipoamide dehydrogenase component of the pyruvate and 2-oxoglutarate dehydrogenase complexes (the lpd gene product) is not involved in this stimulation. These results are discussed in relation to our recent studies showing a possible involvement of lipoic acid and of the 2-oxoacid dehydrogenases in the binding-protein-dependent transports. [ABSTRACT FROM AUTHOR]