Interaction of gonadotropin‐releasing hormone and its agonist analogs with Ca2+ in a nonpolar milieu. Correlation with biopotencies

Extracellular Ca2+ is necessary for the action of gonadotropin-releasing hormone (GnRH). Assuming that this partly because of the interaction of the hormone with the relatively abundant extracellular Ca2+ in the low dielectric milieu of the bilayer plasma membrane, we studied the interaction of GnRH and five of its agonist analogs with Ca2+ under membrane-mimetic conditions. The peptides used, in increasing order of their reported gonadotropin-releasing activities, were: des-amide GnRH (or GnRH-OH); [Ala6]GnRH; [D-Ala6]GnRH; des-Gly10[D-Ala6,Pro9-NHEt]GnRH and, des-Gly10[D-Trp6,Pro9-NHEt]GnRH. Changes in the far-UV CD and fluorescence spectra of these peptides in trifluoroethanol were used to monitor conformational changes and obtain the Ca2+-binding isotherms. The data show that GnRH and its active analogs contain two Ca2+ binding sites, whereas the inactive analogs have only one. The extent of Ca2+ binding by the agonist peptides paralleled their biological potency ranking. The superactive analog des-Gly10[D-Trp6,Pro9-NHEt]GnRH exhibited the ability to transport Ca2+ ions across large unilamellar vesicles of dimyristoylphosphatidylcholine. Our study shows that significant differences among the GnRH and its analog peptides, suggestive of differences in their conformations, are manifested only in the presence of Ca2+. This observation would provide a basis for understanding GnRH action in terms of the hormone's interaction with Ca2+ in the lipid milieu.