Oka Y, Czech M P
J Biol Chem. 1984 Jul 10;259(13):8125-33.
Irradiation of intact rat adipocytes with high intensity ultraviolet light in the presence of 0.5 microM [3H] cytochalasin B results in the labeling of Mr 43,000 and 46,000 proteins that reside in the plasma membrane fraction. In contrast to the Mr 46,000 protein, the Mr 43,000 component is not observed in the microsome fraction and exhibits lower affinity for [3H]cytochalasin B. Photolabeling of the Mr 43,000 protein is inhibited by cytochalasin D, indicating it is not a hexose transporter component. The Mr 46,000 protein exhibits characteristics expected for the glucose transporter such that D-glucose or 3-O-methylglucose but not cytochalasin D inhibits its photolabeling with [3H] cytochalasin B. Furthermore, insulin addition to intact cells either prior to or after photoaffinity labeling of the Mr 46,000 protein causes a redistribution of this component from the low density microsomes to the plasma membrane fraction, as expected for the hexose transporter. Photolabeling of transporters in both the low density microsome and plasma membrane fractions is inhibited when intact cells are equilibrated with 50 mM ethylidene glucose prior to irradiation with [3H]cytochalasin B. Incubation of intact cells with 50 mM ethylidene glucose for 1 min at 15 degrees C leads to an intracellular concentration of only 2 mM. Under these conditions, the photoaffinity labeling in intact cells of hexose transporters that fractionate with the low density microsomes is unaffected, indicating these transporters are not exposed to the extracellular medium. In contrast, photolabeling in intact insulin-treated cells of hexose transporters that fractionate with the plasma membrane is inhibited under these incubation conditions. The results demonstrate that insulin action results in the exposure to the extracellular medium of previously sequestered hexose transporters.
在0.5微摩尔[3H]细胞松弛素B存在的情况下，用高强度紫外光照射完整的大鼠脂肪细胞，会使位于质膜部分的分子量为43,000和46,000的蛋白质被标记。与分子量为46,000的蛋白质不同，分子量为43,000的组分在微粒体部分未被观察到，并且对[3H]细胞松弛素B的亲和力较低。细胞松弛素D可抑制分子量为43,000的蛋白质的光标记，表明它不是己糖转运蛋白的组分。分子量为46,000的蛋白质表现出葡萄糖转运蛋白所预期的特征，即D-葡萄糖或3-O-甲基葡萄糖可抑制其与[3H]细胞松弛素B的光标记，但细胞松弛素D则不能。此外，在对分子量为46,000的蛋白质进行光亲和标记之前或之后，向完整细胞中添加胰岛素会导致该组分从低密度微粒体重新分布到质膜部分，这与己糖转运蛋白的情况预期一致。当完整细胞在用[3H]细胞松弛素B照射之前用50毫摩尔乙叉葡萄糖平衡时，低密度微粒体和质膜部分的转运蛋白的光标记均受到抑制。在15摄氏度下，将完整细胞与50毫摩尔乙叉葡萄糖孵育1分钟，细胞内浓度仅为2毫摩尔。在这些条件下，与低密度微粒体分离的己糖转运蛋白在完整细胞中的光亲和标记不受影响，表明这些转运蛋白未暴露于细胞外介质。相反，在这些孵育条件下，与质膜分离的己糖转运蛋白在完整胰岛素处理细胞中的光标记受到抑制。结果表明，胰岛素作用导致先前被隔离的己糖转运蛋白暴露于细胞外介质。
