Interaction of substance P and its N- and C-terminal fragments with Ca2+: implications for hormone action.
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 microM 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.(ABSTRACT TRUNCATED AT 250 WORDS)
D000595:Amino Acid Sequence; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D066298:In Vitro Techniques; D007700:Kinetics; D008081:Liposomes; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D018040:Receptors, Neurokinin-1; D017981:Receptors, Neurotransmitter / Q000378:metabolism; D013373:Substance P / Q000737:chemistry* / Q000378:metabolism
D008081:Liposomes; D010446:Peptide Fragments; D018040:Receptors, Neurokinin-1; D017981:Receptors, Neurotransmitter; D013373:Substance P; D002118:Calcium