IDENTIFICATION AND CHARACTERIZATION OF THE GLUCOSE-TRANSPORT PROTEIN OF THE BOVINE BLOOD-BRAIN-BARRIER
IDENTIFICATION AND CHARACTERIZATION OF THE GLUCOSE-TRANSPORT PROTEIN OF THE BOVINE BLOOD-BRAIN-BARRIER
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DOI：
10.1042/bj2470101
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发表时间：
1987-10-01
影响因子：
4.1
通讯作者：
BALDWIN, SA
中科院分区：
文献类型：
--
作者：
KASANICKI, MA;CAIRNS, MT;BALDWIN, SA
来自牛大脑皮层微血管的葡萄糖转运蛋白已被鉴定并表征其结合配体[4-3H]细胞松弛素b的能力。微血管膜被发现含有一组葡萄糖抑制的高亲和力细胞松弛素b结合位点[113 .+-]。16 (S.E.M.) pmol/mg膜蛋白]，关联常数为6.8 +-。1.8 (S.E.M.).mu.M-1。d -葡萄糖以31 mM的Ki抑制了这些位点的结合。通过[4-3H]细胞松弛素B的光亲和标记鉴定了该转运蛋白，并发现该转运蛋白在SDS/聚丙烯酰胺凝胶上以明显Mr 55,000的宽带迁移。d -葡萄糖抑制标记，而l -葡萄糖不抑制标记。内糖苷酶F处理产生了明显Mr为46,000的更清晰的条带，表明转运蛋白被糖基化。然而，与人红细胞葡萄糖转运体相反，用endo- β。-半乳糖苷酶对微血管蛋白的电泳迁移率影响不大。光标记蛋白的胰蛋白酶消化产生表观MQr 18000的放射性片段，类似于消化标记的人红细胞葡萄糖转运蛋白产生的片段。此外，将一种与光标记转运蛋白相同的Mr蛋白，通过抗血清在微血管膜的Western blots上标记为完整的红细胞转运蛋白，并针对其N端和c端区域对应的合成肽。结果表明，牛脑皮质微血管内皮细胞葡萄糖转运蛋白与人红细胞葡萄糖转运蛋白具有结构同源性。
The glucose-transport protein from bovine cerebral-cortex microvessels has been identified and characterized by virtue of its ability to bind the ligand [4-3H]cytochalasin B. Microvessel membranes were found to contain a single set of glucose-inhibitable high-affinity cytochalasin B-binding sites [113 .+-. 16 (S.E.M.) pmol/mg of membrane protein], with an association constant of 6.8 .+-. 1.8 (S.E.M.) .mu.M-1. D-Glucose inhibited the binding to these sites with a Ki of 31 mM. The transport protein was identified by photoaffinity labelling with [4-3H]cytochalasin B and was found to migrate as a broad band of apparent Mr 55,000 on SDS/polyacrylamide gels. Labelling was inhibited by D-glucose, but not by L-glucose. Treatment with endoglycosidase F yielded a sharper band of apparent Mr 46,000, indicating that the transport protein is glycosylated. However, in contrast with the human erythrocyte glucose transporter, digestion with endo-.beta.-galactosidase had little effect on the electrophoretic mobility of the microvessel protein. Tryptic digestion of the photolabelled protein yielded a radioactive fragment of apparent MQr 18,000, similar to that of the fragment produced by digestion of the labelled human erythrocyte glucose transporter. In addition, a protein of Mr identical with that of the photolabelled transporter was labelled on Western blots of microvessel membranes by antisera raised against the intact erythrocyte transporter and against synthetic peptides corresponding to its N- and C-terminal regions. It is concluded that the glucose-transport protein of bovine cerebral-cortex microvessel endothelial cells shows structural homology with the human erythrocyte glucose transporter.