Racher K I, Culham D E, Wood J M
Department of Microbiology and Guelph-Waterloo Centre for Graduate Work in Chemistry and Biochemistry, University of Guelph, Guelph, Ontario N1G 2W1, Canada.
Biochemistry. 2001 Jun 19;40(24):7324-33. doi: 10.1021/bi002331u.
Transporter ProP of Escherichia coli, a solute-H+ symporter, can sense and respond to osmotic upshifts imposed on cells, on membrane vesicles, or on proteoliposomes that incorporate purified ProP-(His)6. In this study, proline uptake catalyzed by ProP was used as a measure of its osmotic activation, and the requirements for osmosensing were defined using the proteoliposome system. The initial rate of proline uptake increased with decreasing external pH and increasing DeltaPsi, lumen negative. Osmotic upshifts increased DeltaPsi by concentrating lumenal K+, but osmotic activation of ProP could be distinguished from this effect. Osmotic activation of ProP resulted from changes in Vmax, though osmotic shifts also increased the KM for proline. Osmotic activation could be described as a reversible, osmotic upshift-dependent transition linking (at least) two transporter protein conformations. No correlation was observed between ProP activation and the position of the anions of activating sodium salts within the Hofmeister series of solutes. Both the magnitude of the osmotic upshift required to activate ProP and the ProP activity attained were similar for membrane-impermeant osmolytes, including NaCl, glucose, and PEG 600. The membrane-permeant osmolytes glycerol, urea, PEG 62, and PEG 106 failed to activate ProP. Two poly(ethylene glycol)s, PEG 150 and PEG 200, were membrane-permeant and did not cause liposome shrinkage, but they did partially activate ProP-(His)6.
大肠杆菌的转运蛋白ProP是一种溶质-H⁺同向转运体，它能够感知并响应施加于细胞、膜囊泡或含有纯化的ProP-(His)6的蛋白脂质体上的渗透压升高。在本研究中，由ProP催化的脯氨酸摄取被用作其渗透激活的一种度量，并且使用蛋白脂质体系统确定了渗透感应的要求。脯氨酸摄取的初始速率随着外部pH值的降低和膜电位差（内腔为负）的增加而增加。渗透压升高通过使内腔K⁺浓缩来增加膜电位差，但ProP的渗透激活可以与这种效应区分开来。ProP的渗透激活是由Vmax的变化引起的，尽管渗透压变化也增加了脯氨酸的KM值。渗透激活可以被描述为一种可逆的、依赖于渗透压升高的转变，它连接（至少）两种转运蛋白构象。在ProP激活与激活钠盐的阴离子在霍夫迈斯特系列溶质中的位置之间未观察到相关性。对于包括NaCl、葡萄糖和PEG 600在内的膜不透性渗透剂而言，激活ProP所需的渗透压升高幅度以及达到的ProP活性是相似的。膜可透性渗透剂甘油、尿素、PEG 62和PEG 106未能激活ProP。两种聚乙二醇PEG-150和PEG-200是膜可透性的，并且不会导致脂质体收缩，但它们确实部分激活了ProP-(His)6。
