Simpson I A, Vannucci S J, DeJoseph M R, Hawkins R A
Department of Neuroscience , Milton S. Hershey Medical Center, Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033, USA.
J Biol Chem. 2001 Apr 20;276(16):12725-9. doi: 10.1074/jbc.M010897200. Epub 2001 Jan 12.
The transport of glucose across the mammalian blood-brain barrier is mediated by the GLUT1 glucose transporter, which is concentrated in the endothelial cells of the cerebral microvessels. Several studies supported an asymmetric distribution of GLUT1 protein between the luminal and abluminal membranes (1:4) with a significant proportion of intracellular transporters. In this study we investigated the activity and concentration of GLUT1 in isolated luminal and abluminal membrane fractions of bovine brain endothelial cells. Glucose transport activity and glucose transporter concentration, as determined by cytochalasin B binding, were 2-fold greater in the luminal than in the abluminal membranes. In contrast, Western blot analysis using a rabbit polyclonal antibody raised against the C-terminal 20 amino acids of GLUT1 indicated a 1:5 luminal:abluminal distribution. Western blot analysis with antibodies raised against either the intracellular loop of GLUT1 or the purified erythrocyte protein exhibited luminal:abluminal ratios of 1:1. A similar ratio was observed when the luminal and abluminal fractions were exposed to the 2-N-4(3)Hbenzoxyl-1,3-bis-(d-mannos-4-yloxyl)-2-propylamine ([(3)H]ATB-BMPA) photoaffinity label. These observations suggest that either an additional glucose transporter isoform is present in the luminal membrane of the bovine blood-brain barrier or the C-terminal epitope of GLUT1 is "masked" in the luminal membrane but not in the abluminal membranes.
葡萄糖跨哺乳动物血脑屏障的转运由GLUT1葡萄糖转运蛋白介导，该蛋白集中在脑微血管的内皮细胞中。多项研究支持GLUT1蛋白在管腔膜和基膜之间呈不对称分布（1:4），且细胞内转运蛋白占相当比例。在本研究中，我们调查了牛脑内皮细胞分离的管腔膜和基膜部分中GLUT1的活性和浓度。通过细胞松弛素B结合测定，葡萄糖转运活性和葡萄糖转运蛋白浓度在管腔膜中比在基膜中高2倍。相比之下，使用针对GLUT1 C末端20个氨基酸产生的兔多克隆抗体进行的蛋白质印迹分析表明管腔膜与基膜的分布比例为1:5。用针对GLUT1细胞内环或纯化的红细胞蛋白产生的抗体进行蛋白质印迹分析，显示管腔膜与基膜的比例为1:1。当管腔膜和基膜部分暴露于2-N-4(3)H苯并氧基-1,3-双-(d-甘露糖-4-氧基)-2-丙胺([(3)H]ATB-BMPA)光亲和标记时，观察到类似的比例。这些观察结果表明，要么牛血脑屏障的管腔膜中存在另一种葡萄糖转运蛋白同工型，要么GLUT1的C末端表位在管腔膜中被“掩盖”，而在基膜中则没有。
