Calcium binding and translocation properties of glucagon and its fragments

Abstract

Earlier studies have indicated that the N- and C-terminal regions of glucagon are functionally and structurally different. We have sought to understand this distinction in terms of the interaction of glucagon and its N- and C-terminal fragments with Ca2+, Mg2+, and Zn2+ in a nonpolar milieu. CD spectral data, in 98% (v/v) trifluoroethanol in water, reveal two binding sites for Ca2+ and Mg2+ and one site for Zn2+ in the intact hormone as well as in the C-terminal 19-29 fragment. The 1-6 fragment did not bind Zn2+ and formed a 2:1 peptide-Ca2+ or -Mg2+ complex. With glucagon and the 19-29 fragment, cation binding caused changes in the peptide's helix content. Fluorescence spectral changes involving Trp-25 in the 19-29 fragment and Trp-25 and Tyr-10 and/or Tyr-13 in glucagon were seen on Ca2+ binding to one of the two sites, while Zn2+ binding produced no change in fluorescence. The spectral data suggest that Ca2+ and Zn2+ binding sites (with Kd in the micromolar range in 98% trifluoroethanol) are distinct and are contained in the C-terminal domain of glucagon. Glucagon and the 19-29 fragment, but not the 1-6 fragment, caused an influx of Ca2+ (as monitored by spectral changes in arsenazo III) in unilamellar vesicles made of dimyristoyllecithin. Leakage of vesicle contents induced by the 19-29 fragment was minimal but was significant (approximately 10%) in the case of glucagon. The transport data suggest an interaction of the C-terminal domain of glucagon with Ca2+ at the lipid-water interface.(ABSTRACT TRUNCATED AT 250 WORDS)

Glucagon receptors

“…In an acute hyperglucagonemic state, as internalized endosomal GRs are recycled back to the cell surface with changes in glucagon-binding activity in plasma membrane completely reversed in less than 2 h [23], a recycling pathway may also apply to the endosome-associated miniglucagon [140]. In support of this, using circular dichroism and fluorescence methods, it has been shown that the Ca 2+ -binding capacity of glucagon is maintained in the glucagon-(19-29) fragment, and that both peptides display some ability to penetrate the lipid bilayer in a hydrophobic environment with the bound Ca 2+ ion [141]. Thus, potential translocation of the bioactive glucagon- (19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29) fragment across the endosomal membrane may be conceivable.…”

Endosomal Proteolysis of Glucagon at Neutral pH Generates the Bioactive Degradation Product Miniglucagon-(19–29)

“…In support of this hypothesis, biochemical assays have shown that plasma membranes were able to fuse with early endosomes containing previously internalized ligands (43). Interestingly, using circular dichroism and fluorescence spectroscopy, it has been shown that the Ca 2ϩ -binding capacity of glucagon is maintained in the (19 -29) fragment, and that both peptides display some ability to penetrate the lipid bilayer in a hydrophobic environment with the bound Ca 2ϩ ion (44). Thus, translocation of the (19 -29) bioactive glucagon fragment across the endosomal membrane, comparable to that observed for bacterial and plant toxins (45), cannot be formally excluded.…”

Three-dimensional structure of Phyllomedusin, a NK1 receptor agonist bound to dodecylphosphocholine micelles

“…These results demonstrate a specific interaction of Phyllomedusin with calcium in TFE. It is of interest to note that a similar situation exists in case of SP, a tachykinin and two other peptide hormones -insulin and glucagon Ananthanarayanan et al, 1997;Brimble and Ananthanarayanan, 1992;Brimble and Ananthanarayanan, 1993;Qi et al, 2000). This indicates that specific constraints may be imposed on the Phyllomedusin structure on its interaction with calcium at low concentrations, which may play a role in the bioactive conformation of the peptide and its interaction with the receptor.…”