The role of functional sulfhydryl groups in active transport in Escherichia coli membrane vesicles

Hr, Kaback; Patel, Lekha

doi:10.1021/bi00602a010

Cited by 37 publications

(18 citation statements)

References 36 publications

(47 reference statements)

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“…This indicates that a proportion of the GalP sites have been inactivated by NEM in a similar manner to that described for Lacy [25]. In addition, it has been reported elsewhere that NEM does not affect the magnitude of the proton motive force generated in membrane vesicles oxidizing ascorbate-PMS [26].…”

Section: Resultssupporting

confidence: 77%

Glucose protection against [¹⁴C] N‐ethylmaleimide labelling of a protein in galactose‐transporting membrane vesicles of Escherichia coli

Kaethner

Horne

1980

FEBS Letters

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Section: Resultssupporting

confidence: 77%

Glucose protection against [¹⁴C] N‐ethylmaleimide labelling of a protein in galactose‐transporting membrane vesicles of Escherichia coli

Kaethner

Horne

1980

FEBS Letters

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“…6 and 7). Inverted vesicles prepared from cells expressing either of these mutants generated a stable electrochemical gradient (inside positive and acid) in the presence of ascorbate and phenazine methosulfate (30), as demonstrated by fluorescence spectroscopy using ⌬ -and ⌬pH-sensitive dyes (data not shown). Under these conditions chloramphenicol is actively transported into the vesicles (15).…”

Section: Effect Of the Substrates On [ 14 C]nem Labeling Of Single-mentioning

confidence: 91%

Determinants of Substrate Recognition by the Escherichia coli Multidrug Transporter MdfA Identified on Both Sides of the Membrane

Adler

Bibi

2004

Journal of Biological Chemistry

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The Escherichia coli multidrug transporter MdfA contains a membrane-embedded charged residue (Glu-26) that was shown to play an important role in substrate recognition. To identify additional determinants of multidrug recognition we isolated 58 intragenic second-site mutations that restored the function of inactive MdfA E26X mutants. In addition, two single-site mutations that enhanced the activity of wild-type MdfA were identified. Most of the mutations were found in two regions, the cytoplasmic half of transmembrane segments (TMs) 4, 5, and 6 (cluster 1) and the periplasmic half of TM 1 and 2 (cluster 2). The identified residues were mutated to cysteines in the background of a functional cysteineless MdfA, and substrate protection against alkylation was analyzed. The results support the suggestion that the two clusters are involved in substrate recognition. Using inverted membrane vesicles we observed that a proton electrochemical gradient (⌬˜H؉, inside positive and acidic) enhanced the substrate-protective effect in the cytoplasmic region, whereas it largely reduced this effect in the periplasmic side of MdfA. Therefore, we propose that substrates interact with two sites in MdfA, one in the cytoplasmic leaflet of the membrane and the other in the periplasmic leaflet. Theoretically, these domains could constitute a large part of the multidrug pathway through MdfA.

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“…It is well known that Nethylmaleimide reacts at the binding site of the lac carrier protein (24,25). Gal-S-Gal protects the lac transport system against sulfhydryl inactivation (24,25,27), and treatment of vesicles with sulfhydryl reagents blocks NphGal binding (22) and decreases the Vmax of lactose transport without altering the apparent Km. Taken together, the observations suggest that DEPC (17) and regenerates counterflow activity in DEPC-treated vesicles (Fig.…”

Section: Methodsmentioning

confidence: 99%

Effect of diethylpyrocarbonate on lactose/proton symport in Escherichia coli membrane vesicles.

Padan¹,

Patel²,

Kaback³

1979

Proc. Natl. Acad. Sci. U.S.A.

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Exposure of Escherichia coli ML 308-225 membrane vesicles to the histidine-specific reagent diethylpyrocarbonate (DEPC) led to concentration-and time-dependent inactivation of active lactose transport, and the sensitivity of the system to inactivation was enhanced when an electrochemical proton gradient (AiH+, interior negative and alkaline) was generated across the vesicle membrane. Although P-D-galactopyranosyl 1-thio-t-D-galactopyranoside blocked DEPC The chemosmotic hypothesis of Mitchell (1-3) proposes that energy derived from respiration, photochemical reactions, or ATP hydrolysis can be transformed into a transmembrane electrochemical gradient of protons (AOH+) that is the immediate driving force for many active transport systems in bacterial cells. More specifically, the hypothesis states that substrate-specific membrane proteins (i.e., porters, carriers, or permeases) mediate the coupling between AH+ and substrate accumulation by catalyzing the translocation of substrate with protons, the substrate moving against and the protons with their respective electrochemical gradients (i.e., symport).Cytoplasmic membrane vesicles prepared from Escherichia coli, which retain the same polarity and configuration as the membrane in the intact cell (4-6) as well as the capacity to convert respiratory energy into a 4AH+ (interior negative and alkaline) (7), catalyze the active transport of many substrates, including 3-galactosides (8). Moreover, studies with intact cells (9) and this in vitro system (7,(10)(11)(12)(13)(14) provide virtually unequivocal evidence for the central obligatory role of chemosmotic phenomena in active transport. Recent evidence with isolated membrane vesicles (15, 16) indicates that carriermediated lactose efflux down a concentration gradient is an ordered mechanism in which lactose is released first from the carrier, followed by loss of a proton, and that the unloaded carrier may be negatively charged. In MATERIALS AND METHODSGrowth of Cells and Preparation of Membrane Vesicles. E. coli ML 308-225 (lac-, lacZ-, lacY+, lacA +) and ML 30 (lad +, lacZ +, lacY +, lacA +) were grown on minimal medium A containing 1.0% disodium succinate (hexahydrate), and membrane vesicles were prepared as described (19,20).Treatment with DEPC. Vesicles suspended in 100 mM potassium phosphate (pH 6.6) were titrated to pH 6.0 with 100 mM monobasic potassium phosphate and adjusted to a final concentration of about 2.0 mg of protein per ml by adding appropriate volumes of 100 mM potassium phosphate (pH 6.0). Small portions of 2.0 M DEPC (freshly prepared in absolute ethanol) were added to the membrane suspensions such that the final concentration of ethanol was never more than 0.5% (at concentrations up to 1.0%, ethanol has no significant effect on any of the activities studied). Immediately after addition of DEPC, the suspensions were vigorously agitated to achieve complete mixing and incubated at room temperature on a magnetic stirrer for given periods of time. Reactions were terminated by 1:5 dilution ...

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The role of functional sulfhydryl groups in active transport in Escherichia coli membrane vesicles

Cited by 37 publications

References 36 publications

Glucose protection against [¹⁴C] N‐ethylmaleimide labelling of a protein in galactose‐transporting membrane vesicles of Escherichia coli

Glucose protection against [¹⁴C] N‐ethylmaleimide labelling of a protein in galactose‐transporting membrane vesicles of Escherichia coli

Determinants of Substrate Recognition by the Escherichia coli Multidrug Transporter MdfA Identified on Both Sides of the Membrane

Effect of diethylpyrocarbonate on lactose/proton symport in Escherichia coli membrane vesicles.

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The role of functional sulfhydryl groups in active transport in Escherichia coli membrane vesicles

Cited by 37 publications

References 36 publications

Glucose protection against [14C] N‐ethylmaleimide labelling of a protein in galactose‐transporting membrane vesicles of Escherichia coli

Glucose protection against [14C] N‐ethylmaleimide labelling of a protein in galactose‐transporting membrane vesicles of Escherichia coli

Determinants of Substrate Recognition by the Escherichia coli Multidrug Transporter MdfA Identified on Both Sides of the Membrane

Effect of diethylpyrocarbonate on lactose/proton symport in Escherichia coli membrane vesicles.

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Glucose protection against [¹⁴C] N‐ethylmaleimide labelling of a protein in galactose‐transporting membrane vesicles of Escherichia coli

Glucose protection against [¹⁴C] N‐ethylmaleimide labelling of a protein in galactose‐transporting membrane vesicles of Escherichia coli