AFFINITY LABELING OF THE ADIOPCYTE HEXOSE CARRIER

主要重点将放在故意和非故意的用法上 己糖载体在大鼠脂肪细胞和人体内的亲和标记 红血球。在前几年，有几个潜在有用的衍生品 都是用糖作为亲和配基。这个 这些衍生品的初步测试将通过以下方式完成 它们要么对细胞有意义，要么对细胞没有意义，它们抑制了 一旦运输不可逆转地结合到载体部位，即其行为 被D-葡萄糖和其他底物类似物抑制，并且它们结合 载体作为运输抑制的线性函数。少校 被测试的衍生物将是一种细胞修饰的碳-6葡萄糖 具有芳基叠氮作为偶联剂的衍生物和一种不具代表性的 具有芳基叠氮或马来酰亚胺偶联的双(甘露糖)衍生物 一群人。这些衍生物标记己糖载体的能力 在质膜中或在细胞内脂肪细胞的情况下 然后将对细胞器进行测试。在脂肪细胞中，将特别关注 注意胰岛素的作用，这是为了使细胞内移位 载体进入质膜。同样重要的是要确定 不同条件下的己糖载体。使用一个或多个运营商标识 这些标记中的更多将通过使用单克隆来确认 或针对红细胞己糖载体制备的多克隆抗体。当它发生的时候 已经确定这些衍生品可以在外部标记 在整个细胞或内部载体中处置的载体， 对易位过程进行了详细分析。特别强调的是 考虑到运营商回收进出手机的可能性 表面，这种回收现象可能是更普遍的 脂肪细胞的膜蛋白周转过程。以前的研究基于 关于己糖在红细胞中的转运可能受以下因素调节的研究 ATP的枯竭也将延长。这些研究将探索 载体或调节蛋白被磷酸化的可能性和 因此在这些细胞中被激活。这样的机构可以提供一种替代方案 激活己糖转运的手段。这项工作的主要意义 将证明一个有功能的膜蛋白可以亲和 在红细胞和胰岛素敏感的脂肪细胞中标记，以及这样的 亲和标记法可用于监测膜的处置 蛋白质在各种刺激或抑制的条件下。

The major emphasis will be on the use of both permeant and impermeant affinity labels for the hexose carrier in rat adipocytes and human erythrocytes. In the previous years several potentially useful derivatives were developed, all of which use a sugar as the affinity ligand. The initial testing of these derivatives will be completed by demonstrating that they either are permeant or impermeant to cells, that they inhibit transport irreversibly upon binding to the carrier site, that their action is inhibited by D-glucose and other substrate analogues, and that they bind the carrier as a linear function of transport inhibition. The major derivatives to be tested will be a cell-permeant carbon-6 modified glucose derivative having an aryl azide as the coupling agent, and an impermeant bis(mannose) derivative having either an aryl azide or maleimide coupling group. The ability of these derivatives to label the hexose carrier either in the plasma membrane or in the case of adipocytes in intracellular organelles will then be tested. In adipocytes particular attention will be paid to the effects of insulin, which are to translocate intracellular carriers to the plasma membrane. It will also be important to identify the hexose carrier under various conditions. Carrier identity using one or more of these labels will be confirmed with the use of either a monoclonal or polyclonal antibody made to the erythrocyte hexose carrier. When it has been established that these derivatives can label either externally disposed carrier in whole cells or internal carriers, the kinetics of the translocation process will be analyzed in detail. Particular emphasis will be paid to the possibility that carriers recycle to and from the cell surface, and that this recycling phenomenon may be part of a more general process of membrane protein turnover in adipocytes. Previous studies based on the finding that hexose transport in erythrocytes may be regulated by ATP depletion will also be extended. These studies will explore the possibility that the carrier or a regulatory protein is phosphorylated and thus activated in these cells. Such a mechanism may provide an alternate means for activating hexose transport. The major significance of this work will be to demonstrate that a functional membrane protein can be affinity labeled in erythrocytes and in insulin sensitive adipocytes and that such affinity labeling can be used to monitor the disposition of membrane proteins under various conditions of stimulation or inhibition.