Effect of membrane potential on the kinetic parameters of the Na+ or H+ melibiose symport in Escherichia coli membrane vesicles

ABSTRACT The membrane-permeabilizing ability of the Escherichia coli enterotoxin STb was evaluated using brush border membrane vesicles isolated from piglet jejunum and a membrane-potential-sensitive fluorescent probe, 3,3′-dipropylthiadicarbocyanine iodide. A strong membrane potential was generated by the efflux of K+ ions from the vesicles in the presence of the potassium ionophore valinomycin. Under these conditions, preincubation of the vesicles with STb efficiently depolarized the membrane in a dose-dependent and saturable manner. This activity was independent of pH, however, at least between pH 5.5 and 8.0. On the other hand, in the absence of valinomycin, STb had no significant influence on the measured fluorescence levels, indicating that it was unable to modify the ionic selectivity of the intact membrane. In agreement with the fact that the integrity of the disulfide bridges of STb is known to be essential for its biological activity, a reduced and alkylated form of the toxin was unable to depolarize the membrane in the presence of valinomycin. Furthermore, two previously described poorly active STb mutants, M42S and K22A-K23A, showed no membrane-permeabilizing capacity. These results demonstrate for the first time that STb can permeabilize its target membrane and suggest that it does so by forming nonspecific pores.

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Mechanism of proline transport in Escherichia coli K12. I. Effect of a membrane potential on the kinetics of 2H+/proline symport in cytoplasmic membrane vesicles.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(17)42863-8 ◽

1984 ◽

Vol 259 (12) ◽

pp. 7791-7796 ◽

Cited By ~ 4

Author(s):

T Mogi ◽

Y Anraku

Keyword(s):

Escherichia Coli ◽

Membrane Potential ◽

Cytoplasmic Membrane ◽

Membrane Vesicles ◽

Proline Transport ◽

Escherichia Coli K12 ◽

Kinetics Of

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Mechanisms of active transport in isolated bacterial membrane vesicles. 28. Membrane potential and active transport in membrane vesicles from Escherichia coli

Biochemistry ◽

10.1021/bi00696a011 ◽

1975 ◽

Vol 14 (25) ◽

pp. 5451-5461 ◽

Cited By ~ 232

Author(s):

Shimon Schuldiner ◽

H. Ronald Kaback

Keyword(s):

Escherichia Coli ◽

Membrane Potential ◽

Active Transport ◽

Membrane Vesicles ◽

Bacterial Membrane

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Adenosine 5'-triphosphate synthesis energized by an artificially imposed membrane potential in membrane vesicles of Escherichia coli.

Journal of Bacteriology ◽

10.1128/jb.127.1.154-161.1976 ◽

1976 ◽

Vol 127 (1) ◽

pp. 154-161 ◽

Cited By ~ 15

Author(s):

T Tsuchiya ◽

B P Rosen

Keyword(s):

Escherichia Coli ◽

Membrane Potential ◽

Membrane Vesicles

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Evidence for an electrogenic 3-deoxy-2-oxo-d-gluconate–proton co-transport driven by the protonmotive force in Escherichia coli K12

Biochemical Journal ◽

10.1042/bj1680211 ◽

1977 ◽

Vol 168 (2) ◽

pp. 211-221 ◽

Cited By ~ 7

Author(s):

A Lagarde

Keyword(s):

Escherichia Coli ◽

Membrane Potential ◽

Membrane Vesicles ◽

Ph Gradient ◽

Protonmotive Force ◽

Chemical Gradient ◽

Escherichia Coli K12 ◽

Relative Contribution ◽

Isolated Membrane Vesicles ◽

External Ph

Evidence is presented indicating that the carrier-mediated uptake of 3-deoxy-2-oxo-D-gluconate and D-glucuronate in Escherichia coli K12 is driven by the deltapH and deltapsi components of the protonmotive force. 1. Approximately two protons enter the cells with each sugar molecule, independent of the sugar and the strain used. 2. In respiring cells, the magnitude of the pH gradient alone, as measured by distribution of [3H]acetate, appears to be insufficient to account for the chemical gradient of 3-deoxy-2-oxo-D-gluconate that is developed between pH 6.0 and 8.0. 3. If the external pH is varied between 5.5 and 8.0, 3-deoxy-2-oxo-D-gluconate uptake is gradually inhibited by valinomycin plus K+ ions, whereas the inhibition caused by nigericin is concomitantly relieved, thus reflecting the relative contribution of deltapH and deltapsi to the total protonmotive force at each external pH. 4. 3-Deoxy-2-oxo-D-gluconate can be transiently accumulated into isolated membrane vesicles in response to an artificially induced pH gradient. The process is stimulated when the membrane potential is collapsed by valinomycin in the presence of K+ ions.

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