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Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1979 Dec;76(12):6221–6225. doi: 10.1073/pnas.76.12.6221

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

E Padan, L Patel, H R Kaback
PMCID: PMC411835  PMID: 392515

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 (delta- muH+, interior negative and alkaline) was generated across the vesicle membrane. Although beta-D-galactopyranosyl 1-thio-beta-D-galactopyranoside blocked DEPC inactivation, binding of p-nitrophenyl alpha-D-galactopyranoside was not significantly altered, indicating that DEPC does not react at the binding sites of the lac carrier protein. Strikingly, vesicles treated with DEPC exhibited an increased apparent Km for delta- muH+-driven lactose transport and counterflow but no change in the Vmax of these reactions and no change in the apparent Km or Vmax of facilitated diffusion. Moreover, DEPC treatment increased the apparent Km observed for delta- muH+-driven proline and D-lactate transport with no change in Vmax. Finally, the lactose counterflow activity of DEPC-treated vesicles was regenerated by subsequent exposure to hydroxylamine. It is suggested that a histidyl residue(s) in the lac carrier or another protein in the translocation complex is involved either in the binding and translocation of protons or in a conformational change that may occur upon protonation of the lac carrier protein.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

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