Protein Science, Vol 1, Issue 7 884-891, Copyright © 1992 by Cold Spring Harbor Laboratory Press

ARTICLE

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

M. SHOEMAKER, P. C. LIN and B. HALEY
Division of Medicinal Chemistry & Pharmaceutics, College of Pharmacy and the Markey Cancer Center, University of Kentucky Medical Center, 800 Rose Street, Lexington, Kentucky 40536-0093

Glucagon, a peptide hormone synthesized and secreted by {alpha} islet cells, regulates glucose homeostasis by several mechanisms. Using [{gamma}(32)P]8N(3)GTP, 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 {mu}M with either [{alpha}(32)P]8N(3)GTP or [{gamma}(32)P]8N(3)GTP. GTP protected photolabeling with an apparent k(d) of 15 {mu}M, whereas ATP was less effective as a protector, exhibiting an apparent k(d) of about 30 {mu}M. 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. Mg(2+) at 150 {mu}M enhanced photoinsertion (twofold), whereas at 2-10 mM, it inhibited photoinsertion. Both Ca(2+) and Zn(2+) 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 [(32)P]8N(3)GTP. 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.
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