Affinity Labeling of Purine Nucleotide Sites in Proteins

嘌呤核苷酸在细胞内具有多功能作用， 新陈代谢. 例如，他们直接参与 激酶和ATP酶反应，它们作为调节剂起作用， 许多别构酶，它们参与（通过 环腺苷酸的作用）在几种激素的作用中， 它们有助于血小板的聚集， 参与脱氢酶反应， 辅酶分子 本研究的直接目的是 开发工具，以便系统地探讨 嘌呤核苷酸位点的氨基酸残基 proteins. 亲和标记的一般方法将是 基于先前在本发明中合成的试剂使用 实验室以及新的试剂，其中包括，在 嘌呤或核糖环的不同位置，烷基化 能够与蛋白质共价反应的试剂; 因此嘌呤核苷酸部分将提供特异性 用于结合内的蛋白质的化学修饰 网站. 这些核苷酸烷化剂对 兔肌肉丙酮酸激酶以及大鼠肝脏 将检查外膜ATP酶。 血液的聚集 血小板是由ADP诱导的，可能是通过与 血小板表面膜上的受体位点。 亲和力 用嘌呤核苷酸类似物标记将用于 血小板ADP受体蛋白的鉴定和分离 膜。 此外，反应性cAMP衍生物的作用 对血小板cAMP磷酸二酯酶的影响。 为 每种酶，亲和标记反应的动力学 将检查，试剂掺入的范围将是 测定和放射性肽将被分离， 蛋白水解酶的标记酶，以确定 改性残留物。 第一类核苷酸类似物 也就是我们描述过的氟磺酰基苯甲酰基核苷 已经被用来在特定的地点反应， 50蛋白质;很可能，我们所使用的新化合物 开发将同样具有广泛的适用性， 标记蛋白质中的核苷酸位点， 研究将作为使用这些模型的原型， 化合物来探测其他酶。

Purine nucleotides have a multi-functional role in cellular metabolism. For example, they are directly involved in kinase and ATPase reactions, they functin as regulators of many allosteric enzymes, they are involved (through the effects of cyclic AMP) in the action of several hormones, they contribute to the aggregation of platelets, and they participate in dehydrogenase reactions as part of the coenzyme molecule. The immediate aim of this study is to develop the tools which will allow systematic exploration of the amino acid residues in the purine nucleotide sites of proteins. The general approach of affinity labeling will be used based on reagents previously synthesized in this laboratory as well as on new reagents which incorporate, at various positions of the purine or ribose ring, alkylating agents capable of reacting covalently with the protein; the purine nucleotide moiety will thus provide the specificity for chemical modification of the proteins within the binding sites. The effects of these nucleotide alkylating agents on the rabbit muscle pyruvate kinase as well as on the rat liver ecto-ATPase will be examined. The aggregation of blood platelets is induced by ADP, presumably by binding to receptor sites on the platelet surface membrane. Affinity labeling with the purine nucleotide analogues will be used to identify and isolate the ADP receptor protein of platelet membranes. Further, the effects of reactive cAMP derivatives on the platelet cAMP phosphodiesterase will be explored. For each enzyme, the kinetics of the affinity labeling reaction will be examined, the extend of reagent incorporation will be determined and radioactive peptides will be isolated from proteolytic digests of labeled enzyme to identify the modified residue. The first class of nucleotide analogues which we described, the fluorosulfonylbenzoyl nucleosides, has already been used to react at specific sites of more than 50 proteins; it is likely that the newer compounds which we develop will similarly have broad applicability in the labeling of nucleotide sites in proteins and that these studies will serve as prototypes for the use of these compounds to probe other enzymes.