Arbuckle M I, Kane S, Porter L M, Seatter M J, Gould G W
Division of Biochemistry and Molecular Biology, University of Glasgow, Scotland.
Biochemistry. 1996 Dec 24;35(51):16519-27. doi: 10.1021/bi962210n.
The liver-type (GLUT2) and brain-type (GLUT3) human facilitative glucose transporters exhibit distinct kinetics (K(m) values for deoxyglucose transport of 11.2 +/- 1.1 and 1.4 +/- 0.06 mM, respectively) and patterns of substrate transport (GLUT2 is capable of D-fructose transport, GLUT3 is not) [Gould, G. W., Thomas, H. M., Jess, T. J., & Bell, G. I. (1991) Biochemistry 30, 5139-5145]. We have generated a range of chimeric glucose transporters composed of regions of GLUT2 and GLUT3 with a view to identifying the regions of the transporter which are involved in substrate recognition and binding. The functional characteristics of these chimeras were determined by expression in Xenopus oocytes after microinjection of cRNA. Replacement of the region from the start of putative transmembrane helix 7 to the C-terminus of GLUT3 with the corresponding region from GLUT2 results in a chimera with the ability to transport fructose and exhibits a K(m) for 2-deoxyglucose transport of close to that observed for wild-type GLUT2 (8.3 +/- 0.3 mM compared to 11.2 +/- 1.1 mM). Replacement of the region in GLUT3 from the end of helix 7 to the C-terminus with the corresponding region from GLUT2 resulted in a species which was unable to transport fructose and whose K(m) for 2-deoxyglucose was indistinguishable from wild-type GLUT3. We have determined the affinity for 2-deoxyglucose, D-fructose, and D-galactose of these and other chimeras. In addition, the Ki for maltose, a competitive inhibitor of 2-deoxyglucose transport, which binds to the exofacial sugar binding site was determined for these chimeras. The results obtained support a model in which the seventh putative transmembrane-spanning helix is intimately involved in the selection of transported substrate and in which this region plays an important role in determining the K(m) for 2-deoxyglucose. Additional data is presented which suggests that a region between the end of putative transmembrane helix 7 and the end of helix 10, together with sequences in the N-terminal half of the protein may also participate in substrate recognition and transport catalysis.
肝脏型（GLUT2）和脑型（GLUT3）人易化葡萄糖转运蛋白表现出不同的动力学特征（脱氧葡萄糖转运的K(m)值分别为11.2±1.1和1.4±0.06 mM）以及底物转运模式（GLUT2能够转运D-果糖，GLUT3则不能）[古尔德，G.W.，托马斯，H.M.，杰斯，T.J.，&贝尔，G.I.（1991年）《生物化学》30，5139 - 5145]。我们构建了一系列由GLUT2和GLUT3区域组成的嵌合葡萄糖转运蛋白，旨在确定转运蛋白中参与底物识别和结合的区域。通过显微注射cRNA后在非洲爪蟾卵母细胞中表达来确定这些嵌合体的功能特性。用GLUT2的相应区域替换从假定跨膜螺旋7起始处到GLUT3 C末端的区域，会产生一种具有转运果糖能力的嵌合体，其2 - 脱氧葡萄糖转运的K(m)值接近野生型GLUT2所观察到的值（8.3±0.3 mM，而野生型GLUT2为11.2±1.1 mM）。用GLUT2的相应区域替换GLUT3中从螺旋7末端到C末端的区域，得到的一种物质无法转运果糖，其2 - 脱氧葡萄糖的K(m)值与野生型GLUT3无明显差异。我们已经确定了这些嵌合体以及其他嵌合体对2 - 脱氧葡萄糖、D - 果糖和D - 半乳糖的亲和力。此外，还测定了这些嵌合体对麦芽糖（一种与2 - 脱氧葡萄糖转运竞争的抑制剂，它结合在胞外糖结合位点）的抑制常数（Ki）。所获得的结果支持这样一种模型，即第七个假定的跨膜螺旋密切参与所转运底物的选择，并且该区域在确定2 - 脱氧葡萄糖的K(m)值方面起重要作用。还提供了其他数据，表明在假定跨膜螺旋7末端和螺旋10末端之间的区域，连同蛋白质N端一半的序列，也可能参与底物识别和转运催化。
