Walmsley A R, Martin G E, Henderson P J
Krebs Institute for Biomolecular Research, University of Sheffield, Western Bank, United Kingdom.
J Biol Chem. 1994 Jun 24;269(25):17009-19.
The binding of sugars and antibiotics to the overexpressed D-galactose-H+ symport protein (GalP) can be monitored from changes in the fluorescence of 8-anilino-1-naphthalenesulfonate (ANS) equilibrated with inside-out vesicles. Transported sugars, such as D-glucose and D-galactose, cause an enhancement in the ANS fluorescence of up to 13%. Nontransported sugars that have little, if any, affinity for GalP, such as L-galactose and L-glucose, have no effect upon the ANS fluorescence. Cytochalasin B and forskolin, which are potent inhibitors of the transporter, produce little change in the fluorescence, but are capable of reversing the D-galactose/D-glucose enhancement in fluorescence. Sugars that bind to GalP but are not transported, such as methyl-beta-D-glucose, produce only a slight quench in the ANS fluorescence, but again reverse the enhancement in fluorescence induced by transported sugars. A simple interpretation is that the increase in ANS fluorescence is attributable to the sugar-induced reorientation of the transporter from an inward- to an outward-facing conformation. Nontransported sugars and antibiotics, which are thought to bind at the inner membrane face of the transporter, are able to reverse the fluorescence enhancement by binding to the inward-facing conformation. The postulated reorientation process was sufficiently slow to follow its progress by stopped-flow fluorometry. The Kd for the binding of D-galactose to the inward-facing site was 7.22 (+/- 1.49) mM, and the rate constants for outward and inward reorientation of the transporter were 4.06 (+/- 0.16) s-1 and 1.36 (+/- 0.18) s-1, respectively. The overall Kd values for a range of sugars and antibiotics have been determined, and the involvement of each sugar hydroxyl group in the recognition and translocation processes has been assessed.
糖和抗生素与过表达的D-半乳糖-H⁺同向转运蛋白（GalP）的结合可通过与内翻囊泡平衡的8-苯胺基-1-萘磺酸盐（ANS）荧光变化来监测。转运的糖，如D-葡萄糖和D-半乳糖，可使ANS荧光增强高达13%。对GalP亲和力很小（如果有的话）的非转运糖，如L-半乳糖和L-葡萄糖，对ANS荧光没有影响。细胞松弛素B和福斯可林是该转运体的有效抑制剂，它们对荧光的影响很小，但能够逆转D-半乳糖/D-葡萄糖引起的荧光增强。与GalP结合但不被转运的糖，如甲基-β-D-葡萄糖，只会使ANS荧光略有淬灭，但同样能逆转转运糖诱导的荧光增强。一种简单的解释是，ANS荧光的增加归因于糖诱导转运体从内向构象向外向构象的重新定向。非转运糖和抗生素被认为在内膜表面与转运体结合，它们能够通过与内向构象结合来逆转荧光增强。假定的重新定向过程足够缓慢，可以通过停流荧光法跟踪其进程。D-半乳糖与内向位点结合的解离常数（Kd）为7.22（±1.49）mM，转运体向外和向内重新定向的速率常数分别为4.06（±0.16）s⁻¹和1.36（±0.18）s⁻¹。已经确定了一系列糖和抗生素的总体Kd值，并评估了每个糖羟基在识别和转运过程中的作用。
