Affinity Labeling of Purine Nucleotide Sites in Proteins

嘌呤核苷酸在细胞代谢中具有多种功能。例如，它们直接参与激酶和三磷酸腺苷酶的反应，它们作为许多变张酶的调节剂起作用，它们（通过环AMP的作用）参与几种激素的作用，它们有助于血小板的聚集，它们作为辅酶分子的一部分参与脱氢酶的反应。本研究的直接目的是开发一种工具，可以系统地探索蛋白质嘌呤核苷酸位点上的氨基酸残基。亲和标记的一般方法将基于本实验室先前合成的试剂，以及在嘌呤或核糖环的不同位置加入能够与蛋白质共价反应的烷基化剂的新试剂；嘌呤核苷酸片段因此将为结合位点内蛋白质的化学修饰提供特异性。这些核苷酸烷基化剂对兔肌丙酮酸激酶和大鼠肝外三磷酸腺苷酶的影响将被研究。血小板的聚集是由ADP诱导的，可能是通过与血小板表面膜上的受体位点结合而引起的。嘌呤核苷酸类似物的亲和标记将用于鉴定和分离血小板膜的ADP受体蛋白。此外，还将探讨活性cAMP衍生物对血小板cAMP磷酸二酯酶的影响。对于每种酶，将检查亲和标记反应的动力学，确定试剂掺入的范围，并从标记酶的蛋白水解酶中分离放射性肽以鉴定修饰残基。我们描述的第一类核苷酸类似物，氟磺酰基苯甲酰核苷，已经被用于在50多种蛋白质的特定位点发生反应；我们开发的新化合物很可能同样在标记蛋白质中的核苷酸位点方面具有广泛的适用性，并且这些研究将作为使用这些化合物探测其他酶的原型。

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.