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

Abstract

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 ...

Site-specific mutagenesis of histidine residues in the lac permease of Escherichia coli.

“…The results presented here provide strong confirmation of previous studies (23)(24)(25) demonstrating that acylation of right-side-out membrane vesicles with diethylpyrocarbonate or photooxidation in the presence of rose bengal inactivates lactose/H' symport via the lac permease. Since these operations are known to be relatively specific for histidine residues and subsequent studies (25) with purified, reconstituted lac permease show that rose bengal-catalyzed photooxidation modifies two of the four histidine residues in the permease, it was concluded that histidine residues are critical for symport.…”

“…Although the mechanism by which the lac permease transduces A!iH+ into a lactose concentration gradient is unknown, studies (23)(24)(25) with diethylpyrocarbonate and rose bengal in right-side-out membrane vesicles suggest that histidine residues may play an important role in coupling fl+ and lactose translocation. Site-directed mutagenesis (26) provides a means of altering amino acid residues in a protein with a high degree of specificity, and the approach has been used recently (27,28) to evaluate the role of cysteine-148 in the permease.…”

Sugar specificity and sugar-proton interaction for the hexose-proton-symport system of Chlorella

“…The previously published data that a strong decrease of affinity happens above pH 7.5 [7] therefore do not reflect the pH-dependence of the transport system per se. It had been postulated previously that a histidine group participates in the transport of hexose by Chlorella [35] and of P-galactoside by E. coli [36] with the imidazole nitrogen providing the binding site for proton.…”

His-65 in the proton–sucrose symporter is an essential amino acid whose modification with site-directed mutagenesis increases transport activity

“…For example, the H322R mutation of LacY lost protonmotive-forcecoupling, and the H376Q mutation of LacS resulted in complete loss of transport function, whereas in AtSUC1 H65Q and H65R mutations lead to equal or higher transport rates than that of the wild-type symporter. Moreover, the DEPCsensitive histidine(s) of lactose permease is not directly involved in substrate binding (31). In contrast, substrate protection experiments have shown that His-65 is at, or at least conformationally linked to, the substrate-binding site of the sucrose symporter (ref.…”