9423108 Colman Nucleotides have a multi-functional role in cellular metabolism. For example, they are directly involved in kinase involved in kinase reactions, they participate in dehydrogenase reactions as part of the coenzyme molecule, they function as regulators of many allosteric enzymes, they are involved (through the effects of cyclic AMP) in the action of several hormones, and they contribute to the aggregation of platelets. The aim of this study is to develop the tools which will allow a systematic exploration of the amino acid residues in the 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 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 adenylosuccinate synthetase, as well as on adenylosuccinate lyase, will be examined. Further, the reactions of cAMP derivatives with platelet cAMP phosphodiesterases will be explored. 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 characterize the ADP receptor protein of platelet membranes. For each protein, the kinetics of the affinity labeling reaction will be examined, the extent of reagent incorporation will be determined and radioactive peptides will be isolated from proteolytic digests of labeled enzyme to identify the modified residue. The catalytic and ligand binding properties as well as the conformational characteristics of the modified enzyme will be evaluated. 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. In addition, these nucleotide analogues will have applications in the biotechnology industry where they can be used to detect expression of cloned nucleotide binding proteins, and to provide the knowledge base for rational design of drugs functioning as specific inhibitors targeted to particular nucleotide-requiring enzymes. %%% Nucleotides have a multifunctional role in cellular metabolism. For example, nucleotides participate directly in numerous enzymatic reactions, they function as activatos or inhibitors of many regulatory enzymes, and the"second messenger" cyclic AMP carries out the action of several hormones. The aim of this study is to develop the tools which will allow a systematic exploration of the region of an enzyme which binds nucleotides. The general approach of "affinity labeling" will be used in which we attach to a natural nucleotide a reactive portion (called an alkylating agent) which is capable of forming a permanent link to a protein. The nucleotide moiety of the new reagent will direct the compound to a specific binding site on the protein, and the reactive portion will make the binding irreversible so that the binding site can be isolated and characterized. We will now study the effects of these nucleotide alkylating agents ontwo enzymes ctitical for purine nucleotide biiosynthesis and on an enzyme important in the degradation of the regulatory nucleotide cyclic AMP. The first class of nucleotide analogues which we described, the fluorosulfonylbenzoyl nucleosides, has aleady 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 b road applicability in the lab eling of nucleotide sites in proteins and that these studies will serve as prototypes for the use of these compounds to probe other enzymes. In addition, these nucleotide analogues will hve applications in the biotechnology industry where they can be used to detect expression of cloned nucleotide binding proteins, and to provide the knowledge base for rational design of drugs functioning as specific inhibitors targeted to particular nucleotide-requiring enzymes. ***
