Evidence for monomeric and oligomeric hormone-binding domains in affinity-purified gonadotropin receptor from rat ovary.
Rat ovarian lutropin/choriogonadotropin receptor was purified from a Triton X-100-solubilized membrane preparation by affinity chromatography with Affi-Gel 10 coupled to purified human choriogonadotropin. The affinity-purified receptor preparations contained a single class of high-affinity binding sites for 125I-labeled human choriogonadotropin, with an equilibrium dissociation constant (Kd) of 2.5 x 10(-9) M, which is comparable to the Kd values for membrane-bound and solubilized receptors. The purified receptor appeared as two dominant bands with molecular weights of 135,000 and 92,000 after sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS/PAGE) under nonreducing conditions. These two bands were also detected in subsequent direct ligand blotting analysis when the purified receptor was electrophoretically transferred to a nitrocellulose membrane after SDS/PAGE under nonreducing conditions. When the individual affinity-purified receptor bands were electroeluted from the gel and analyzed again by SDS/PAGE under nonreducing conditions, both the Mr 92,000 and the 135,000 proteins retained their original molecular form even when 8 M urea was included in the gel. However, when the electrophoretically purified Mr 92,000 and 135,000 bands were subjected to SDS/PAGE under reducing conditions, the Mr 135,000 species was almost completely converted to a Mr 92,000 band, but the Mr 92,000 species did not undergo any alteration in molecular weight. The results suggest that the lutropin/choriogonadotropin receptor from rat ovary exists in two molecular forms, and the higher molecular weight form appears to be composed of disulfide-linked Mr 92,000 subunit, which comprises the hormone-binding domain.
Zhang
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QY
Department of Obstetrics/Gynecology, University of Michigan, Ann Arbor 48109-0278.
Menon
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D000818:Animals; D001665:Binding Sites; D002462:Cell Membrane / Q000378:metabolism; D006063:Chorionic Gonadotropin / Q000378:metabolism*; D002846:Chromatography, Affinity; D003902:Detergents; D005260:Female; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D017830:Octoxynol; D010053:Ovary / Q000378:metabolism*; D011092:Polyethylene Glycols; D051381:Rats; D011919:Rats, Inbred Strains; D011967:Receptors, Gonadotropin / Q000302:isolation & purification / Q000378:metabolism*
D006063:Chorionic Gonadotropin; D003902:Detergents; D046911:Macromolecular Substances; D011967:Receptors, Gonadotropin; D011092:Polyethylene Glycols; D017830:Octoxynol