Purified reconstituted lac carrier protein from Escherichia coli is fully functional.
Purified reconstituted lac carrier protein from Escherichia coli is fully functional.
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DOI：
10.1073/pnas.81.6.1629
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发表时间：
1984-03
影响因子：
11.1
通讯作者：
Paul V. Viitanen;Garcia Ml;Kaback Hr
中科院分区：
文献类型：
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作者：
Paul V. Viitanen;Garcia Ml;Kaback Hr
从大肠杆菌质膜中提纯的Lac载体蛋白重组的蛋白脂质体催化每一种典型的β-半乳糖苷转运系统的易位反应(即主动转运、逆流、促进内流和外流)，其周转次数和表观Km值与右侧外翻膜泡中观察到的相似。此外，详细的动力学研究表明，重组体系表现出与膜泡中观察到的类似的性质。施加膜电位(psi，内部负值)会导致表观Km显著降低(降低7到10倍)，而Vmax增加较小(约等于3倍)。在次极大值处，复合载体呈现两相动力学特征，其中一个组分表现出主动输运的动力学参数，另一个组分表现出促进扩散的特征。最后，在低乳糖浓度下，初始流入速度与质子电化学梯度的平方成线性关系。这一结果为以下论点提供了定量的支持，即一个单一的多肽物种，即lac y基因的产物，负责β-半乳糖苷运输系统中的每一种典型的运输反应。
Proteoliposomes reconstituted with lac carrier protein purified from the plasma membrane of Escherichia coli catalyze each of the translocation reactions typical of the beta-galactoside transport system (i.e., active transport, counterflow, facilitated influx and efflux) with turnover numbers and apparent Km values comparable to those observed in right-side-out membrane vesicles. Furthermore, detailed kinetic studies show that the reconstituted system exhibits properties analogous to those observed in membrane vesicles. Imposition of a membrane potential (delta psi, interior negative) causes a marked decrease in apparent Km (by a factor of 7 to 10) with a smaller increase in Vmax (approximately equal to 3-fold). At submaximal values of delta psi, the reconstituted carrier exhibits biphasic kinetics, with one component manifesting the kinetic parameters of active transport and the other exhibiting the characteristics of facilitated diffusion. Finally, at low lactose concentrations, the initial velocity of influx varies linearly with the square of the proton electro-chemical gradient. The results provide quantitative support for the contention that a single polypeptide species, the product of the lac y gene, is responsible for each of the transport reactions typical of the beta-galactoside transport system.