Determination of the effect of acetylation of specific lysine residues in human growth hormone on its affinity for somatogenic receptors by an affinity selection technique.
A technique is described to study the effect of acetylation of individual lysine residues in peptide hormones on the affinity for their receptors, and is illustrated for the case of human growth hormone (hGH) binding to somatogenic receptors. The hGH was partially acetylated with high specific activity [3H]-acetic anhydride and the product ([3H]-Ac-hGH) was incubated with solubilised affinity-purified somatogenic receptors (from male rat liver) in the presence and absence of excess unlabelled hGH. The receptor-bound and unbound labelled hormone were separated by gel filtration and subjected to HPLC tryptic peptide mapping after the addition of cold carrier Ac-hGH. Peaks of [3H] radioactivity were assigned to peptides corresponding to the acetylation of specific lysine residues in the hGH sequence by amino acid analysis and sequencing. Comparison of the relative intensities of corresponding [3H] peaks in the peptide maps of added receptor, bound and unbound [3H]-Ac-hGH, enabled the relative receptor-binding potencies of different acetylated hGH species to be determined. Acetylation of lysine 168 or 172 in hGH greatly decreases its receptor-binding affinity, acetylation of lysine 115 probably causes a minor decrease, whereas acetylation of lysines 38, 70, and the N-terminal amino group have no appreciable effect. Acetylation of lysine 140 causes a significant increase in receptor-binding affinity.
D000107:Acetylation; D000818:Animals; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D013006:Growth Hormone / Q000031:analogs & derivatives / Q000378:metabolism*; D006801:Humans; D008099:Liver / Q000032:analysis; D008239:Lysine / Q000378:metabolism*; D008297:Male; D010446:Peptide Fragments / Q000378:metabolism; D051381:Rats; D011986:Receptors, Somatotropin / Q000378:metabolism*; D014357:Trypsin
D010446:Peptide Fragments; D011986:Receptors, Somatotropin; D013006:Growth Hormone; D014357:Trypsin; D008239:Lysine