ISSN:
0006-3525
Keywords:
Chemistry
;
Polymer and Materials Science
Source:
Wiley InterScience Backfile Collection 1832-2000
Topics:
Chemistry and Pharmacology
Notes:
In an attempt to understand the role of Ca2+ on the bioactive conformation of peptide hormones, we have examined the interaction between Ca2+ and the neuropeptide substance P. Using CD spectroscopy to monitor conformational changes caused by Ca2+ binding, we found no significant binding of the cation by substance P in water. However, a substantial conformational change occurred in the hormone on Ca2+ addition in trifluoroethanol or an 80:20 (v/v) mixture of acetonitrile and trifluoroethanol. A biphasic binding of Ca2+ was observed in these solvents with saturation at 2 cations per hormone molecule. Mg2+ caused a relatively smaller conformational change in the hormone. A peptide corresponding to residues 1-7 at the N-terminal fragment of substance P showed a weak nonsaturating binding of Ca2+ in the nonpolar solvents whereas the 7-11 C-terminal fragment peptide displayed a binding indicative of an 1 : 1 Ca2+ / peptide complex. Ca2+ binding by the hormone and the 7-11 fragment was also monitored by changes in fluorescence of the phenylalanyl residues. The results support the conclusion drawn from the CD data about a distinct Ca2+ binding site in the C-terminal part of substance P. The Kd values obtained from fluorescence data were 160 µM for Ca2+ and 1 mM for Mg2+ binding by substance P. The hormone and the two peptide fragments were also tested for their effect on the stability of dimyristoyl lecithin vesicles. Substance P and the N-terminal fragment caused no significant leakage of either fluorescent dyes or K+ trapped in the vesicles. Nor did they cause membrane fusion as monitored by the fluorescence quenching method. In contrast, the C-terminal 7-11 fragment caused considerable leakage of vesicular contents and promoted membrane fusion. When tested for their ability to translocate Ca2+ across the lipid bilayer in dimyristoyl lecithin vesicles containing the Ca2+ indicator Arsenazo III, both substance P and the 7-11 fragment showed significant Ca2+ transport while the 1-7 fragment showed negligible transport. The apparent transport of the 7-11 fragment is attributable to its effect on the integrity of the bilayer membrane. These data distinguish the N- and C-terminal domains of substance P in terms of their lipid-dependent interaction with Ca2+. In light of the requirement of extracellular Ca2+ for substance P action, the data are also suggestive of the possibility of the involvement of Ca2+ in the bioactive conformation of this hormone and in its interaction with the receptor. © 1992 John Wiley & Sons, Inc.
Additional Material:
7 Ill.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1002/bip.360321217
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