Johnson Zachary Lee, Lee Seok-Yong
Department of Biochemistry, Duke University Medical Center, Durham, North Carolina, USA.
Department of Biochemistry, Duke University Medical Center, Durham, North Carolina, USA.
Methods Enzymol. 2015;556:373-83. doi: 10.1016/bs.mie.2014.11.048. Epub 2015 Mar 20.
Secondary active transporters are responsible for the cellular uptake of many biologically important molecules, including neurotransmitters, nutrients, and drugs. Because of their physiological and clinical importance, a method for assessing their transport activity in vitro is necessary to gain a better understanding of how these transporters function at the molecular level. In this chapter, we describe a protocol for reconstituting the concentrative nucleoside transporter from Vibrio cholerae into proteoliposomes. We then describe a radiolabeled substrate uptake assay that can be used to functionally characterize the transporter. These methods are relatively common and can be applied to other secondary active transporters, with or without some modification.
次级主动转运蛋白负责细胞摄取许多具有重要生物学意义的分子，包括神经递质、营养物质和药物。由于它们在生理和临床上的重要性，有必要采用一种体外评估其转运活性的方法，以便更好地了解这些转运蛋白在分子水平上的功能。在本章中，我们描述了一种将霍乱弧菌的浓缩核苷转运蛋白重组到蛋白脂质体中的方案。然后，我们描述了一种放射性标记底物摄取试验，可用于对该转运蛋白进行功能表征。这些方法相对常见，经过或不经过一些修改，均可应用于其他次级主动转运蛋白。
