Calcium binding and translocation properties of glucagon and its fragments.
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)
Brimble
K S
KS
Department of Biochemistry, McMaster University, Hamilton, Ontario, Canada.
Ananthanarayanan
V S
VS
D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002942:Circular Dichroism; D004355:Drug Stability; D005934:Glucagon / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D008081:Liposomes; D008274:Magnesium / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D014364:Tryptophan; D015032:Zinc / Q000378:metabolism
D008081:Liposomes; D010446:Peptide Fragments; C039265:glucagon (19-29); C036296:glucagon (1-6); D014364:Tryptophan; D005934:Glucagon; D008274:Magnesium; D015032:Zinc; D002118:Calcium