Publications

• Mechanism of melibiose/cation symport of the melibiose permease of Salmonella typhimurium.

  Guan L., Nurva S., Ankeshwarapu S.P.

  The MelB permease of Salmonella typhimurium (MelB-ST) catalyzes the coupled symport of melibiose and Na(+), Li(+), or H(+). In right-side-out membrane vesicles, melibiose efflux is inhibited by an inwardly directed gradient of Na(+) or Li(+) and stimulated by equimolar concentrations of internal a ... >> More

  The MelB permease of Salmonella typhimurium (MelB-ST) catalyzes the coupled symport of melibiose and Na(+), Li(+), or H(+). In right-side-out membrane vesicles, melibiose efflux is inhibited by an inwardly directed gradient of Na(+) or Li(+) and stimulated by equimolar concentrations of internal and external Na(+) or Li(+). Melibiose exchange is faster than efflux in the presence of H(+) or Na(+) and stimulated by an inwardly directed Na(+) gradient. Thus, sugar is released from MelB-ST externally prior to the release of cation in agreement with current models proposed for MelB of Escherichia coli (MelB-EC) and LacY. Although Li(+) stimulates efflux, and an outwardly directed Li(+) gradient increases exchange, it is striking that internal and external Li(+) with no gradient inhibits exchange. Furthermore, Trp → dansyl FRET measurements with a fluorescent sugar (2'-(N-dansyl)aminoalkyl-1-thio-β-D-galactopyranoside) demonstrate that MelB-ST, in the presence of Na(+) or Li(+), exhibits (app)K(d) values of ∼1 mM for melibiose. Na(+) and Li(+) compete for a common binding pocket with activation constants for FRET of ∼1 mM, whereas Rb(+) or Cs(+) exhibits little or no effect. Taken together, the findings indicate that MelB-ST utilizes H(+) in addition to Na(+) and Li(+). FRET studies also show symmetrical emission maximum at ∼500 nm with MelB-ST in the presence of 2'-(N-dansyl)aminoalkyl-1-thio-β-D-galactopyranoside and Na(+), Li(+), or H(+), which implies a relatively homogeneous distribution of conformers of MelB-ST ternary complexes in the membrane. << Less

  J. Biol. Chem. 286:6367-6374(2011) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Melibiose permease of Escherichia coli: large scale purification and evidence that H+, Na+, and Li+ sugar symport is catalyzed by a single polypeptide.

  Pourcher T., Leclercq S., Brandolin G., Leblanc G.

  As much as 20-30 mg of functional recombinant melibiose permease (Mel-6His permease) of Escherichia coli, carrying a carboxy-terminal affinity tag for metallic ions (six successive histidines), can be routinely purified from 10 g of cells (dry weight) by combining nickel chelate affinity chromatog ... >> More

  As much as 20-30 mg of functional recombinant melibiose permease (Mel-6His permease) of Escherichia coli, carrying a carboxy-terminal affinity tag for metallic ions (six successive histidines), can be routinely purified from 10 g of cells (dry weight) by combining nickel chelate affinity chromatography and ion exchange chromatography. Mel-6His permease was constructed by modifying the permease gene (melB) in vitro and then overproduced in cells transformed with multicopy plasmids. The tagged permease was efficiently solubilized in the presence of 3-(laurylamido)-N,N'-dimethylaminopropylamine oxide (LAPAO) and high sodium salt concentration and then selectively adsorbed on a nickel nitrilotriacetic acid (Ni-NTA) affinity resin. After the replacement of LAPAO by n-dodecyl beta-D-maltoside to maintain the activity of the soluble permease in low ionic strength media, the permease-enriched fraction (> 90%) was eluted with 0.1 M imidazole and finally purified to homogeneity (> 99%) using ion exchange chromatography. Determination of the permease N-terminal sequence shows that an initiating methionine is missing and that a Ser-Ile-Ser stretch precedes the postulated primary amino acid sequence. Purified permeases, reconstituted in liposomes, display H(+)-, Na(+)-, or Li(+)-dependent sugar binding and active transport activities similar to those of the native permease in its natural environment, proving that all three modes of symport activity are mediated by one and the same polypeptide. << Less

  Biochemistry 34:4412-4420(1995) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.