Showing papers by "Kathleen C. Flanders published in 1982"

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.

Semisynthetic derivatives of glucagon: (des-His1)N epsilon-acetimidoglucagon and N alpha-Biotinyl-N epsilon-acetimidoglucagon.

Abstract: N epsilon-Acetimidoglucagon to be used for semisynthesis was prepared by reacting glucagon with methyl acetimidate hydrochloride at pH 10.2, favoring acetimidation of the sole epsilon-amino group. N epsilon-Acetimidoglucagon was isolated from the crude acetimidoglucagon mixture by anion-exchange chromatography at pH 9.4, producing a derivative which was identical with native glucagon on isoelectric focusing and which by amino acid analysis had greater than 98% of the lysine blocked. The yield was greater than that obtained when tetrahydrophthalic anhydride was used as a chromatographic handle to remove peptides with unreacted amino groups. N epsilon-Acetimidoglucagon closely resembled native glucagon in its biological activity and binding affinity, eliminating the need for deprotection. Semisynthetic N alpha-biotinyl-N epsilon-acetimidoglucagon, prepared by reacting (N-hydroxysuccinimido)biotin with N epsilon-acetimidoglucagon and purified by cation-exchange chromatography, was homogeneous upon isoelectric focusing (pI = 5.2) and exhibited 1.2% of the binding affinity, 2.4% of the biological potency, and 30% of the maximum activity of the native hormone. Preliminary fluorescence microscopy demonstrated binding of N alpha-biotinyl-N epsilon-acetimidoglucagon to glucagon specific receptors following exposure to fluorescein-labeled avidin. Capping of labeled receptors could be visualized with time. (Des-His1)N epsilon-acetimidoglucagon, prepared via a manual Edman degradation of N epsilon-acetimidoglucagon and isolated by cation-exchange chromatography, was homogeneous upon isoelectric focusing (pI = 5.2). The second residue, serine, has also been removed. Semisynthetic coupling of alternative residues to such derivatives will provide insight into the role of the amino-terminal residues in mediating the biological actions of the hormone.