AFFINITY LABELING OF THE ADIOPCYTE HEXOSE CARRIER

脂肪细胞己糖载体的亲和力标记

• 批准号: 3072517
• 负责人: JAMES M. MAY
• 金额: $ 4.2万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-09-01 至 1988-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3072517
• 关键词: adenosine triphosphate; adipocytes; affinity labeling; carbohydrate transport; chemical synthesis; erythrocytes; glucosamine; glucose analog; glucose transport; hexoses; human tissue; monoclonal antibody

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.

重点将放在渗透剂和非渗透剂的使用 大鼠脂肪细胞和人类中己糖载体的亲和标记物 红细胞 在过去的几年里，一些潜在的有用的衍生物 所有这些都使用糖作为亲和配体。 的 这些衍生物的初步测试将通过证明 它们对细胞是渗透性或非渗透性的， 不可逆地运输后结合到载体网站，他们的行动 被D-葡萄糖和其他底物类似物抑制， 载体作为运输抑制的线性函数。 主要 待测试的衍生物将是细胞渗透性碳-6修饰的葡萄糖 具有芳基叠氮化物作为偶联剂的衍生物和不渗透剂 具有芳基叠氮化物或马来酰亚胺偶联的双（甘露糖）衍生物 组 这些衍生物标记己糖载体的能力 在质膜中或在脂肪细胞的情况下在细胞内 然后将对细胞器进行测试。 在脂肪细胞中，将特别注意 胰岛素的作用是在细胞内 载体到质膜。 还必须确定 在各种条件下的己糖载体。 使用一个或多个 这些标记中的更多将通过使用单克隆抗体来证实。 或针对红细胞己糖载体制备的多克隆抗体。 当它已经 已经确定，这些衍生物可以在外部标记 在整个细胞或内部载体中布置的载体， 详细分析了易位过程。 将特别强调 因为携带者有可能在细胞中循环 表面，这种回收现象可能是一个更普遍的一部分， 脂肪细胞膜蛋白周转的过程。 以前的研究基于 红细胞中的己糖转运可能受 ATP耗竭也将延长。 这些研究将探讨 载体或调节蛋白被磷酸化的可能性， 从而在这些细胞中被激活。 这样的机制可以提供替代方案。 用于激活己糖运输的手段。 这项工作的主要意义 将证明功能性膜蛋白可以是亲和的， 在红细胞和胰岛素敏感脂肪细胞中标记， 亲和标记可用于监测膜的处置 蛋白质在不同的刺激或抑制条件下。