Identification of the guanine binding domain peptide of the GTP-binding site of glucagon.
Glucagon, a peptide hormone synthesized and secreted by alpha islet cells, regulates glucose homeostasis by several mechanisms. Using [gamma 32P]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 microM with either [alpha 32P]8N3GTP or [gamma 32P]8N3GTP. GTP protected photolabeling with an apparent kd of 15 microM, whereas ATP was less effective as a protector, exhibiting an apparent kd of about 30 microM. 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 microM 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.
Shoemaker
M
M
Division of Medicinal Chemistry & Pharmaceutics, College of Pharmacy, University of Kentucky Medical Center, Lexington 40536-0093.
Lin
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Haley
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D000225:Adenine / Q000494:pharmacology; D000227:Adenine Nucleotides / Q000494:pharmacology; D000241:Adenosine / Q000494:pharmacology; D000595:Amino Acid Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D002851:Chromatography, High Pressure Liquid; D002918:Chymotrypsin; D004591:Electrophoresis, Polyacrylamide Gel; D005934:Glucagon / Q000737:chemistry* / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000302:isolation & purification
D000227:Adenine Nucleotides; D010446:Peptide Fragments; D006160:Guanosine Triphosphate; D005934:Glucagon; D002918:Chymotrypsin; D000225:Adenine; D000241:Adenosine