Identification of the guanine binding domain peptide of the GTP‐binding site of glucagon

Glucagon, a peptide hormone synthesized and secreted by α islet cells, regulates glucose homeostasis by several mechanisms. Using [γ³²P]8N₃GTP, a proven photoaffinity probe for GTP, a specific nucleotide binding site on human glucagon was detected that showed preference for GTP. Half‐maximal saturation of photoinsertion into the polypeptide hormone was at 8–12 μM with either [α³²P]8N₃GTP or [γ³²P]8N₃GTP. GTP protected photolabeling with an apparent k_d of 15 μM, whereas ATP was less effective as a protector, exhibiting an apparent k_d of about 30 μM. Maximal protection by GTP and ATP was over 90%. UTP, CTP, GDP, ADP, GMP, AMP, guanosine, adenosine, guanine, and adenine were much less effective protectors, indicating that binding is specific for purine nucleoside triphosphates, particularly GTP. Mg²⁺ at 150 μM enhanced photoinsertion (twofold), whereas at 2–10 mM, it inhibited photoinsertion. Both Ca²⁺ and Zn²⁺ at 0.2 mM decreased photoinsertion about 45%. Purification of chymotryptic and tryptic digests of photolabeled glucagon by reverse‐phase high performance liquid chromatography (HPLC) revealed that the N‐terminal peptide, HSQGTF, was the only peptide region covalently photomodified by [³²P]8N₃GTP. GTP, if present during photolysis, greatly reduced both photoinsertion into glucagon and the amount of radiolabeled peptide recovered on HPLC. The specificity of binding to the N‐terminal region is suggestive of a physiological role for a glucagon‐GTP complex in the mechanism of action of this hormone.