Glucagon carboxyl-terminal derivatives: preparation, purification, and characterization

Abstract

Chemical and enzymatic methods have been used to prepare the following series of seven glucagon derivatives modified in the carboxyl-terminal region important for hormone-receptor binding: [des-Asn28,Thr29](homoserine lactone27)glucagon, [des-Asn28,Thr29](homoserine27)glucagon, (S-methyl-Met27)glucagon, [des-Thr29](S-methyl-Met27)-glucagon, [des-Thr29]glucagon,[des-Asn28,Thr29](S-methyl-Met27)glucagon, and [des-Asn28,Thr29]glucagon. The derivatives were isolated in high yield, extensively purified, and chemically characterized. All were found to be full agonists of native glucagon. Binding affinity was evaluated by displacement of mono[125I]iodoglucagon prepared by new methods. Binding and biological activities closely correlated, indicating that most modifications affected the relative binding affinity and relative biological potency of glucagon to a comparable extent. Circular dichroism measured in dilute acid solution resembled that of native glucagon except for [des-Asn28,Thr29]glucagon which displayed increased alpha helicity (25%). All derivatives formed helical structures in 2-chloro-ethanol, although the amount of helicity induced was not closely correlated with biological activity. Binding and biological activities were not affected by removal of Thr-29, though both were reduced 20-fold when Asn-28 was also removed, irrespective of whether homoserine or native methionine remained at the carboxyl terminus. Lactone formation was associated with a further 5-fold reduction in binding affinity but not in activity. Methylation of Met-27 had essentially the same effect as removing the two carboxyl-terminal residues, although the combined effect of both modifications was greater than 100-fold reduction in binding and activity. These findings provide additional insight concerning glucagon structure-function relationships.

Identification of glucagon receptors in rat brain.

“…This is consistent with the observation that virtually the whole glucagon molecule participates in the hepatic receptor regulation process (20). The rank order of potency of glucagon analogs examined for brain membranes (native glucagon > Ne-acetimidoglucagon > [S-methyl-Met27]glucagon > des-Hisl-Ne-acetimidoglucagon) (Fig.…”

“…Synthetic human pancreatic growth hormone-releasing factor, hpGRF-44, was graciously provided by A. Felix (Hoffmann-La Roche). Glucagon analogs, Ne-acetimidoglucagon (19) and des-Hisl-Neacetimidoglucagon were prepared as described by Flanders et al (19); [S-methyl-Met27]glucagon was prepared as described by England et al (20). 1,3,4,6-Tetrachloro-3,6-diphenylglycouril (IODO-GEN) was purchased from Pierce, and cellulose acetate oxoid membrane filters were from Oxoid (Columbia, MD).…”

“…Purified porcine glucagon was iodinated by using IODO-GEN and 1251-monoiodoglucagon was purified as described by England et al (20) to provide a homogeneous and stable derivative with a specific activity of "=8 x 105 cpm/pmol.…”

“…The binding assay was essentially that of England et al (20). The incubation buffer contained 1 mg of bovine serum albumin per ml, 1 mM dithiothreitol, 30 mM Tris-HCl (pH 7.5).…”

“…N8-acetimidoglucagon, a glucagon derivative having an acetimidyl group at the e-amino group of lysine 12 (19) was 1/10th as effective as native glucagon in competing for 1251-monoiodoglucagon binding (IC50 = 31.6 nM). With des-Hisl-Ne-acetimidoglucagon, prepared by manual Edman degradation of Ne-acetimidoglucagon (19), and [S-methyl-Met27]glucagon (20) half-maximal displacement occurred at 398 nM and 296 nM, respectively. Adenylate Cyclase Activation.…”