AFFINITY LABELING OF THE ADIOPCYTE HEXOSE CARRIER

脂肪细胞己糖载体的亲和力标记

• 批准号: 3072516
• 负责人: JAMES M. MAY
• 金额: $ 5.1万
• 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/3072516
• 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.

主要重点是渗透性和非渗透性的使用 大鼠脂肪细胞和人类脂肪细胞中己糖载体的亲和标记 红细胞。 前几年有几种潜在有用的衍生品 开发的，所有这些都使用糖作为亲和配体。 这 这些衍生物的初步测试将通过证明来完成 它们对于细胞来说要么是渗透性的，要么是非渗透性的，它们抑制 结合到载体站点后不可逆转地运输，他们的行动 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。 主要 待测试的衍生物将是可渗透细胞的碳6修饰葡萄糖 具有芳基叠氮化物作为偶联剂和不渗透性的衍生物 具有芳基叠氮化物或马来酰亚胺偶联的双（甘露糖）衍生物 团体。 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 然后将测试细胞器。 在脂肪细胞中，要特别注意 归因于胰岛素的作用，即易位细胞内 质膜的载体。 确定 各种条件下的己糖载体。 使用一种或多种方式识别运营商身份 更多这些标签将通过使用单克隆抗体来确认 或针对红细胞己糖载体制备的多克隆抗体。 当它有 已经确定这些衍生物可以在外部标记 将载体置于整个细胞或内部载体中，其动力学 下面将详细分析易位过程。 特别强调将 支付运营商回收电池的可能性 表面，这种回收现象可能是更普遍的现象的一部分 脂肪细胞膜蛋白周转的过程。 基于之前的研究 发现红细胞中的己糖转运可能受以下因素调节 ATP 消耗也会延长。 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。 这种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具有亲和力 在红细胞和胰岛素敏感脂肪细胞中进行标记 亲和标记可用于监测膜的处置 蛋白质在各种刺激或抑制条件下。