REGULATION OF PLATELET ADENYLATE CYCLASE BY ADP

这项提议是为了继续进行旨在阐明 ADP产生多种效应的机制 血小板 我们将尝试从生物化学的角度 它介导腺苷酸环化酶的抑制，我们已经研究了 通过动力学和配体结合实验，使用ADP类似物 与受体结合的亲和力比天然的 核苷酸 几个互补的方法来隔离的 将使用受体蛋白，包括光亲和标记，亲和 色谱法和可逆配体结合。 我们将测量 2-methylthioADP对血小板和其他细胞的作用，以及对血小板 膜，并确定这种结合是否受到那些 影响其他激动剂结合的条件， 腺苷酸环化酶通过鸟嘌呤结合转导蛋白 与这种酶的激素控制有关。 结合 2-methylthioADP溶解膜蛋白分离， 将使用非变性条件下的电泳来鉴定 ADP结合位点。 一种新的亲和层析介质将用于 分离那些ADP结合蛋白，这些蛋白具有 受体的 为此，ADP将与不溶性载体偶联 基质通过嘌呤环的2-位上的取代基。 ADP的光亲和类似物，其中可光活化的叠氮基官能团 通过间隔基团连接到ADP分子，也通过 在2位取代，将用于表征ADP 受体在许多分析分离中的行为 系统. 通过这些技术分离的蛋白质将用于受体检测。 其功能是通过重组ADP调节的腺苷酸 环化酶系统 腺苷酸调节受体与 环化酶和受体参与血小板活化的ADP将是 研究了

This proposal is for the continuation of experiments designed to elucidate the mechanism through which ADP brings about its varied effects on platelets. We will attempt to characterize biochemically the receptor which mediates the inhibition of adenylate cyclase, which we have studied by means of kinetics and ligand binding experiments, using ADP analogues that bind to the receptor with higher affinity than the natural nucleotide. Several complimentary approaches to the isolation of the receptor protein will be used, including photoaffinity labeling, affinity chromatography and reversible ligand binding. We will measure the binding of 2-methylthioADP to platelets and to other cells, and to platelet membranes, and determine whether this binding is influenced by those conditions that affect the binding of other agonists that regulate adenylate cyclase through the guanine binding transducer proteins implicated in the hormonal control of this enzyme. Binding of 2-methylthioADP to solubilized membrane proteins isolated by electrophoresis under non-denaturing conditions will be used to identify ADP binding sites. A novel affinity chromatography medium will be used to isolate those ADP binding proteins that have the characteristics of the receptor. For this purpose ADP will be coupled to an insoluble support matrix through substituents at the 2- position of the purine ring. Photoaffinity analogues of ADP in which a photoactivatable azido function is attached through a spacer group to the ADP molecule, also through substitution at the 2- position, will be used to characterize the ADP receptor with respect to its behaviour in a number of analytical separation systems. Proteins isolated by these techniques will be tested for receptor function by their ability to reconstitute an ADP-regulated adenylate cyclase system. The relation between the receptor that regulates adenylate cyclase and the receptor involved in platelet activation by ADP will be investigated.