Matsushita K, Patel L, Gennis R B, Kaback H R
Proc Natl Acad Sci U S A. 1983 Aug;80(16):4889-93. doi: 10.1073/pnas.80.16.4889.
Most active transport across the bacterial cell membrane is driven by a proton electrochemical gradient (delta-muH+, interior negative and alkaline) generated via electron transfer through a membrane-bound respiratory chain. This phenomenon is now reproduced in vitro with proteoliposomes containing only two proteins purified from the membrane of Escherichia coli. An o-type cytochrome oxidase was extracted from membranes of a cytochrome d terminal oxidase mutant with octyl beta-D-glucopyranoside after sequential treatment with urea and cholate and was purified to homogeneity by ion-exchange chromatography. The purified oxidase contains four polypeptides (MrS 66,000, 35,000, 22,000, and 17,000), two b-type cytochromes (b558 and b563), and 16-17 nmol of heme b per mg of protein, and it catalyzes the oxidation of ubiquinol and other electron donors with specific activities 20- to 30-fold higher than crude membranes. The lac carrier protein was purified as described. Proteoliposomes were formed in the presence of the oxidase and lac carrier protein by detergent dilution, followed by freeze-thaw/sonication. The system generates a delta-muH+ (interior negative and alkaline) with ubiquinol as electron donor and the magnitude of delta-muH+ is dependent on the concentration of cytochrome o in the proteoliposomes. Furthermore, the proteoliposomes transport lactose against a concentration gradient to an extent that is commensurate with the magnitude of delta-muH+ generated. The results provide powerful additional support for the "chemiosmotic hypothesis" and demonstrate that purified lac carrier protein retains the ability to function in a physiological manner.
大多数细菌细胞膜上的主动运输是由质子电化学梯度（ΔμH⁺，内部为负且呈碱性）驱动的，该梯度通过膜结合呼吸链的电子传递产生。现在，这种现象在体外通过仅含有从大肠杆菌膜中纯化的两种蛋白质的蛋白脂质体得以重现。一种o型细胞色素氧化酶在用尿素和胆酸盐依次处理后，用辛基-β-D-吡喃葡萄糖苷从细胞色素d末端氧化酶突变体的膜中提取，并通过离子交换色谱法纯化至同质。纯化的氧化酶包含四种多肽（分子量分别为66,000、35,000、22,000和17,000）、两种b型细胞色素（b558和b563），每毫克蛋白质含有16 - 17 nmol的血红素b，它催化泛醇和其他电子供体的氧化，比粗膜的比活性高20至30倍。乳糖载体蛋白按所述方法纯化。通过去污剂稀释，随后进行冻融/超声处理，在氧化酶和乳糖载体蛋白存在的情况下形成蛋白脂质体。该系统以泛醇作为电子供体产生ΔμH⁺（内部为负且呈碱性），并且ΔμH⁺的大小取决于蛋白脂质体中细胞色素o的浓度。此外，蛋白脂质体逆浓度梯度转运乳糖的程度与所产生的ΔμH⁺大小相当。这些结果为“化学渗透假说”提供了有力的额外支持，并证明纯化的乳糖载体蛋白保留了以生理方式发挥功能的能力。
