AFFINITY LABELING OF THE ADIOPCYTE HEXOSE CARRIER

重点将放在permeans和impermeans的用法上

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.