KINETIC ANALYSIS OF TOXIN-RECEPTOR INTERACTIONS

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目及 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 许多关键的生物反应发生在双层膜表面之内或之上。 此类反应的一类重要反应是可溶性配体与其细胞表面受体之间的相互作用。 配体-受体相互作用可以是单价的，如小肽配体的情况，二价的，如许多大肽激素和抗体，或者甚至更高的价。 后一类是许多细菌毒素与其细胞表面受体之间的相互作用。 例如，霍乱毒素进入哺乳动物细胞是通过五聚体 B 亚基与细胞膜中的神经节苷脂 Gm1 的结合介导的。 这种相互作用的显着特征包括单个 B 亚基和 Gm1 碳水化合物部分之间的低亲和力单价相互作用、五聚体 B 亚基与几个膜结合的 Gm1 分子之间的高亲和力多价相互作用，以及双层膜内 Gm1 分子的横向移动性。 我们最近将对固定双层的研究扩展到活细胞中毒素加工的研究。 使用动力学和温度控制的流式细胞术，我们正在研究活细胞中各种内化途径的速率和能力。 这些结果被用来建立和测试细胞内配体加工的模型。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Many critical biological reactions occur in or on bilayer membrane surfaces. An important class of such reactions is the interactions between soluble ligands and their cell-surface receptors. Ligand-receptor interactions may be monovalent, as is the case for small peptide ligands, bivalent, as for many large peptide hormones and antibodies, or of even higher valencies. In the latter class are the interactions between many bacterial toxins and their cell-surface receptors. Cholera toxin entry into mammalian cells, for example is mediated by binding of the pentameric B subunit to ganglioside Gm1 in the cell membrane. Notable features of this interaction include a low affinity monovalent interaction between individual B subunits and the carbohydrate moiety of Gm1, a high affinity multivalent interaction between pentameric B subunit with several membrane-bound Gm1 molecules, and the lateral mobility of the Gm1 molecule within the bilayer membrane. We have recently extended our studies on immobilized bialyers to the study of toxin processing in live cells. Using kinetic and temperature controlled flow cytometry, we are investigating the rates and capacities of various internalization pathways in live cells. These results are being used to build and test models of intracellular ligand processing.