REGULATION OF PLATELET ADENYLATE CYCLASE BY ADP

这项提议是为了继续进行旨在阐明 ADP发挥多种作用的机制 血小板。我们将尝试从生物化学的角度描述受体 它介导了腺苷环化酶的抑制，我们已经研究过了 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高于天然的 核苷酸。几种有益的方法来隔离 将使用受体蛋白，包括光亲和标记、亲和 层析和可逆配体结合。我们将测量绑定 2-甲硫基二磷酸对血小板和其他细胞以及对血小板的作用 膜，并确定这种结合是否受那些 影响调节其他激动剂结合的条件 腺苷环化酶通过鸟嘌呤结合转导蛋白 与这种酶的荷尔蒙控制有关。约束： 2-甲硫基腺苷二磷酸对膜蛋白的增溶作用 非变性条件下的电泳法将用于鉴定 ADP结合位点。一种新型的亲和层析介质将用于 分离具有ADP结合蛋白特征的ADP结合蛋白 受体。为此，ADP将与不可溶的支撑物相结合 通过嘌呤环2-位上的取代基形成基质。 具有光激活叠氮功能的ADP的光亲和类似物 通过间隔基连接到ADP分子上，也通过 2位的替换将被用来描述ADP 受体在一些分析分离中的行为 系统。通过这些技术分离的蛋白质将进行受体测试。 通过它们重建ADP调节的腺苷的能力来发挥作用 循环酶系统。调节腺苷酸的受体之间的关系 环化酶和参与ADP激活血小板的受体将是 调查过了。

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.