Padan E, Patel L, Kaback H R
Proc Natl Acad Sci U S A. 1979 Dec;76(12):6221-5. doi: 10.1073/pnas.76.12.6221.
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.
将大肠杆菌ML 308 - 225膜泡暴露于组氨酸特异性试剂焦碳酸二乙酯（DEPC）会导致活性乳糖转运呈浓度和时间依赖性失活，并且当跨膜泡膜产生电化学质子梯度（δ-μH⁺，内部为负且呈碱性）时，该系统对失活的敏感性增强。尽管β-D-吡喃半乳糖基1-硫代-β-D-吡喃半乳糖苷可阻断DEPC失活，但对硝基苯基α-D-吡喃半乳糖苷的结合没有明显改变，这表明DEPC不会在乳糖载体蛋白的结合位点发生反应。引人注目的是，用DEPC处理的膜泡对δ-μH⁺驱动的乳糖转运和逆向转运表现出表观Km增加，但这些反应的Vmax没有变化，且易化扩散的表观Km或Vmax也没有变化。此外，DEPC处理增加了δ-μH⁺驱动的脯氨酸和D-乳酸转运的表观Km，而Vmax没有变化。最后，通过随后暴露于羟胺可使DEPC处理的膜泡的乳糖逆向转运活性恢复。提示乳糖载体中的一个或多个组氨酸残基或转运复合物中的另一种蛋白质参与质子的结合和转运，或参与乳糖载体蛋白质子化时可能发生的构象变化。
