Wright J K, Overath P
Eur J Biochem. 1984 Feb 1;138(3):497-508. doi: 10.1111/j.1432-1033.1984.tb07944.x.
The lactose carrier, a galactoside:H+ symporter in Escherichia coli, has been purified from cytoplasmic membranes by pre-extraction of the membranes with 5-sulfosalicylate, solubilization in dodecyl-O-beta-D-maltoside, Ecteola-column chromatography, and removal of residual impurities by anti-impurity antibodies. Subsequently, the purified carrier was reincorporated into E. coli phospholipid vesicles. Purification was monitored by tracer N-[3H]ethylmaleimide-labeled carrier and by binding of the substrate p-nitrophenyl-alpha-D-galactopyranoside. All purified carrier molecules were active in substrate binding and the purified protein was at least 95% pure by several criteria. Substrate binding to the purified carrier in detergent micelles and in reconstituted proteoliposomes yielded a stoichiometry close to one molecule substrate bound per polypeptide chain. Large unilamellar proteoliposomes (1-5-micron diameter) were prepared from initially small reconstituted vesicles by freeze-thaw cycles and low-speed centrifugation. These proteoliposomes catalyzed facilitated diffusion and active transport in response to artificially imposed electrochemical proton gradients (delta mu H+) or one of its components (delta psi or delta pH). Comparison of the steady-state level of galactoside accumulation and the nominal value of the driving gradients yielded cotransport stoichiometries up to 0.7 proton/galactoside, suggesting that the carrier protein is the only component required for active galactoside transport. The half-saturation constants for active uptake of lactose (KT = 200 microM) or beta-D-galactosyl-1-thio-beta-D-galactoside (KT = 50-80 microM) by the purified carrier were found to be similar to be similar to those measured in cells or cytoplasmic membrane vesicles. The maximum rate for active transport expressed as a turnover number was similar in proteoliposomes and cytoplasmic membrane vesicles (kcat = 3-4 s-1 for lactose) but considerably smaller than in cells (kcat = 40-60 s-1). Possible reasons for this discrepancy are discussed.
H⁺同向转运体，已通过以下步骤从细胞质膜中纯化出来：先用5 - 磺基水杨酸对膜进行预提取，再用十二烷基 - O - β - D - 麦芽糖苷溶解，经Ecteola柱色谱法分离，并用抗杂质抗体去除残留杂质。随后，将纯化后的载体重新整合到大肠杆菌磷脂囊泡中。通过示踪剂N - [³H]乙基马来酰亚胺标记的载体以及底物对硝基苯基 - α - D - 吡喃半乳糖苷的结合来监测纯化过程。所有纯化后的载体分子在底物结合方面均具有活性，并且根据多项标准，纯化后的蛋白质纯度至少为95%。在去污剂胶束和重组蛋白脂质体中，底物与纯化后的载体结合的化学计量比接近每条多肽链结合一个分子的底物。通过冻融循环和低速离心，从最初较小的重组囊泡制备出大单层蛋白脂质体（直径1 - 5微米）。这些蛋白脂质体响应人为施加的电化学质子梯度（ΔμH⁺）或其组分之一（Δψ或ΔpH）催化易化扩散和主动运输。比较半乳糖苷积累的稳态水平和驱动梯度的标称值，得到的共转运化学计量比高达0.7质子/半乳糖苷，这表明载体蛋白是半乳糖苷主动运输所需的唯一成分。纯化后的载体对乳糖（KT = 200微摩尔）或β - D - 半乳糖基 - 1 - 硫代 - β - D - 半乳糖苷（KT = 50 - 80微摩尔）主动摄取的半饱和常数与在细胞或细胞质膜囊泡中测得的相似。以转换数表示的主动运输最大速率在蛋白脂质体和细胞质膜囊泡中相似（乳糖的kcat = 3 - 4秒⁻¹），但远小于在细胞中的速率（kcat = 40 - 60秒⁻¹）。文中讨论了这种差异的可能原因。
