Photoaffinity labeling of pituitary gonadotropin-releasing hormone receptors in goldfish (Carassius auratus).
Receptors for GnRH were labeled by use of an iodinated (125I) photoreactive GnRH derivative [D-Lys6-azidobenzoyl]-GnRH. This derivative was found to bind to two classes of GnRH binding sites: high-affinity/low-capacity sites and low-affinity/high-capacity sites. The binding affinity of [D-Lys6-azidobenzoyl]-GnRH was found to be greater than that of D-Lys6-GnRH, but lower than a superactive fish GnRH agonist [D-Arg6, Trp7, Leu8, Pro9-NEt]-GnRH (sGnRH-A). Analysis of the photoaffinity-labeled goldfish pituitary GnRH receptors by SDS-PAGE and autoradiography indicated the presence of three labeled proteins displaceable by unlabeled sGnRH-A. The first and the most prominently labeled band was a 71,000-Mr protein, the second a 51,000-Mr protein, and the third a minor band of 130,000 Mr. Displacement characteristics of the 71,000- and 130,000-Mr bands were consistent with those of the low-affinity binding sites; displacement of the iodinated ligand from these proteins was achieved only in the presence of 10(-6) M sGnRH-A. The 51,000-Mr band had characteristics similar to those of the high-affinity site; displacement of the labeled ligand was achieved in the presence of 10(-9) M sGnRH-A. These findings provide for the first time some biochemical characterizations of pituitary GnRH receptors in a nonmammalian vertebrate.
Habibi
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Department of Biological Sciences, University of Calgary, Alberta, Canada.
Peter
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Hazum
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D000345:Affinity Labels / Q000378:metabolism; D000818:Animals; D001386:Azides / Q000378:metabolism; D001667:Binding, Competitive; D005260:Female; D006054:Goldfish / Q000378:metabolism*; D007987:Gonadotropin-Releasing Hormone / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D066298:In Vitro Techniques; D007700:Kinetics; D008297:Male; D008970:Molecular Weight; D010902:Pituitary Gland / Q000187:drug effects / Q000378:metabolism*; D011966:Receptors, LHRH / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*
D000345:Affinity Labels; D001386:Azides; D011966:Receptors, LHRH; D007987:Gonadotropin-Releasing Hormone; C031737:LHRH, N-epsilon-azidobenzoyl-Lys(6)-; C058987:5-oxo-pro-his-trp-ser-tyr-gly-trp-leu-pro-glynh2