Lee S H, Cohen N S, Jacobs A J, Brodie A F
Biochemistry. 1979 May 29;18(11):2232-9. doi: 10.1021/bi00578a015.
Membrane vesicles from Mycobacterium phlei contain carrier proteins for proline, glutamine, and glutamic acid. The transport of proline is Na+ dependent and required substrate oxidation. A proline carrier protein was solubilized from the membrane vesicles by treatment with cholate and Triton X-100. Electron microscopic observation of the detergent-treated membrane vesicles showed that they are closed structures. The detergent-extracted proteins were purified by means of sucrose density gradient centrifugation, followed by gel filtration and isoelectric focusing. A single protein with a molecular weight of 20,000 +/- 1000 was found on polyacrylamide gel electrophoresis. Reconstitution of proline transport was demonstrated when the purified protein was incubated with the detergent-extracted membrane vesicles. This reconstituted transport system was specific for proline and required substrate oxidation and Na+. The purified protein was also incorporated into liposomes, and proline uptake was demonstrated when energy was supplied as a membrane potential introduced by K+ diffusion via valinomycin. The uptake of proline was Na+ dependent and was inhibited by uncoupler or by sulfhydryl reagents.
草分枝杆菌的膜泡含有脯氨酸、谷氨酰胺和谷氨酸的载体蛋白。脯氨酸的转运依赖于Na⁺，且需要底物氧化。用胆酸盐和 Triton X - 100处理膜泡后，一种脯氨酸载体蛋白可从膜泡中溶解出来。对经去污剂处理的膜泡进行电子显微镜观察表明它们是封闭结构。去污剂提取的蛋白质通过蔗糖密度梯度离心，随后进行凝胶过滤和等电聚焦进行纯化。在聚丙烯酰胺凝胶电泳上发现了一种分子量为20,000 ± 1000的单一蛋白质。当将纯化的蛋白质与去污剂提取的膜泡一起孵育时，证明了脯氨酸转运的重建。这种重建的转运系统对脯氨酸具有特异性，需要底物氧化和Na⁺。纯化的蛋白质也被整合到脂质体中，当通过缬氨霉素介导的K⁺扩散引入膜电位作为能量供应时，证明了脯氨酸的摄取。脯氨酸的摄取依赖于Na⁺，并受到解偶联剂或巯基试剂的抑制。
