Henderson P J, Martin G E, McDonald T P, Steel A, Walmsley A R
Department of Biochemistry and Molecular Biology, University of Leeds, UK.
Antonie Van Leeuwenhoek. 1994;65(4):349-58. doi: 10.1007/BF00872218.
GalP is the membrane protein responsible for H(+)-driven uptake of galactose into Escherichia coli. It is suggested to be the bacterial equivalent of the mammalian glucose transporter, GLUT1, since these proteins share sequence homology, recognise and transport similar substrates and are both inhibited by cytochalasin B and forskolin. The successful over-production of GalP to 35-55% of the total inner membrane protein of E. coli has allowed direct physical measurements on isolated membrane preparations. The binding of the antibiotics cytochalasin B and forskolin could be monitored from changes in the inherent fluorescence of GalP, enabling derivation of a kinetic mechanism describing the interaction between the ligands and GalP. The binding of sugars to GalP produces little or no change in the inherent fluorescence of the transporter. However, the binding of transported sugars to GalP produces a large increase in the fluorescence of 8-anilino-1-naphthalene sulphonate (ANS) excited via tryptophan residues. This has allowed a binding step, in addition to two putative translocation steps, to be measured. From all these studies a basic kinetic mechanism for the transport cycle under non-energised conditions has been derived. The case of genetical manipulation of the galP gene in E. coli has been exploited to mutate individual amino acid residues that are predicted to play a critical role in transport activity and/or the recognition of substrates and antibiotics. Investigation of these mutant proteins using the fluorescence measurements should elucidate the role of individual residues in the transport cycle as well as refine the current model.
GalP是一种膜蛋白，负责将氢离子驱动的半乳糖转运到大肠杆菌中。由于这些蛋白质具有序列同源性、识别并转运相似的底物，且均受细胞松弛素B和福斯可林抑制，因此它被认为相当于哺乳动物的葡萄糖转运蛋白GLUT1。GalP在大肠杆菌内膜蛋白总量中成功过量表达至35% - 55%，这使得能够对分离出的膜制剂进行直接物理测量。可以通过监测GalP固有荧光的变化来检测抗生素细胞松弛素B和福斯可林的结合情况，从而推导出描述配体与GalP之间相互作用的动力学机制。糖类与GalP的结合对转运蛋白的固有荧光几乎没有影响或没有影响。然而，被转运糖类与GalP的结合会使通过色氨酸残基激发的8 - 苯胺基 - 1 - 萘磺酸盐（ANS）的荧光大幅增加。这使得除了两个假定的转运步骤外，还能测量一个结合步骤。通过所有这些研究，得出了在非激活条件下转运循环的基本动力学机制。利用大肠杆菌中galP基因的基因操作来突变预测在转运活性和/或底物及抗生素识别中起关键作用的单个氨基酸残基。使用荧光测量对这些突变蛋白进行研究，应能阐明单个残基在转运循环中的作用，并完善当前模型。
