IDENTIFICATION AND CHARACTERIZATION OF THE GLUCOSE TRANSPORTER OF THE BLOOD-BRAIN-BARRIER BY CYTOCHALASIN-B BINDING AND IMMUNOLOGICAL REACTIVITY
IDENTIFICATION AND CHARACTERIZATION OF THE GLUCOSE TRANSPORTER OF THE BLOOD-BRAIN-BARRIER BY CYTOCHALASIN-B BINDING AND IMMUNOLOGICAL REACTIVITY
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DOI：
10.1073/pnas.81.22.7233
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发表时间：
1984-01-01
期刊：
影响因子：
--
通讯作者：
WALKER, DM
中科院分区：
文献类型：
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作者：
DICK, APK;HARIK, SI;WALKER, DM
[3 H]细胞松弛素B被用作配体来鉴定和表征大鼠和猪脑微血管中的葡萄糖转运蛋白。特异性细胞松弛素B结合（定义为被过量（500 mM）D-葡萄糖立体特异性置换的总结合分数）是可饱和的。对这种与脑微血管制备物特异性结合的动力学研究表明Kd为0.65-0.88 μ M，最大结合（Bmax）为60-80 pmol/mg蛋白质。相比之下，大脑皮质颗粒组分的Bmax约为大脑微血管的十分之一。各种己糖在体外取代特异性细胞松弛素B与脑微血管结合的能力与这些己糖在体内穿过血脑屏障的能力密切相关。用细胞松弛素B对脑微血管的葡萄糖转运蛋白进行不可逆的光亲和标记，随后进行溶解和聚丙烯酰胺凝胶电泳，标记了MW约为53，000的多肽。针对人红细胞的葡萄糖转运蛋白制备的抗体也与脑微血管的明胶化制备物上的MW约为53，000的多肽反应。脑微血管富含葡萄糖转运蛋白，其分子量和抗原性与人红细胞和其他哺乳动物组织的葡萄糖转运蛋白相似。
[3H]Cytochalasin B was used as a ligand to identify and characterize the glucose transporter in cerebral microvessels of the rat and the pig. Specific cytochalasin B binding, defined as that fraction of the total binding that is stereospecifically displaced by excess (500 mM) D-glucose, is saturable. Kinetic studies of this specific binding to cerebral microvessel preparations showed a Kd of 0.65-0.88 .mu.M and a maximal binding (Bmax) of 60-80 pmol/mg of protein. In comparison, the Bmax of particulate fractions of the cerebral cortex was about one-tenth that of cerebral microvessels. The ability of various hexoses to displace specific cytochalasin B binding to cerebral microvessels in vitro correlated well with the capability of these hexoses to cross the blood-brain barrier in vivo. Irreversible photoaffinity labeling of the glucose transporter of cerebral microvessels with cytochalasin B followed by solubilization and polyacrylamide gel electrophoresis labeled a polypeptide(s) with MW of about 53,000. Antibodies prepared against the glucose transporter of human erythrocytes also reacted with a polypeptide(s) with a MW of about 53,000 on electrophoresed preparations of cerebral microvessels. Cerebral microvessels are richly endowed with a glucose transporter moiety of similar MW and antigenic characteristics as the glucose transporter of human erythrocytes and other mammalian tissues.