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ATPase反应，它们作为许多变构酶的调节器发挥作用，它们(通过环状AMP的作用)参与几种激素的作用，它们促进血小板的聚集，它们作为辅酶分子的一部分参与脱氢酶反应。这项研究的直接目的是开发工具，使系统地探索蛋白质的嘌呤核苷酸位置上的氨基酸残基。亲和标记的一般方法将基于本实验室以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发生共价反应的烷基化试剂的新试剂；因此，嘌呤核苷酸部分将为结合位置内的蛋白质的化学修饰提供特异性。这些核苷酸烷基化试剂对兔肌肉丙酮酸激酶以及对大鼠肝脏外膜-ATPase的影响将被检测。血小板的聚集是由ADP诱导的，推测是通过与血小板表面膜上的受体结合来实现的。用嘌呤核苷酸类似物亲和标记将用于鉴定和分离血小板膜上的ADP受体蛋白。此外，还将探讨反应性cAMP衍生物对血小板cAMP磷酸二酯酶的影响。对于每个酶，将检查亲和标记反应的动力学，确定试剂掺入的程度，并从标记酶的蛋白水解液中分离放射性多肽以鉴定修饰残基。我们所描述的第一类核苷酸类似物，氟磺酰苯甲酰核苷，已经被用来在50多种蛋白质的特定位置上反应；我们开发的较新的化合物很可能同样在标记蛋白质的核苷酸位置方面具有广泛的适用性，这些研究将作为使用这些化合物来探索其他酶的原型。

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.