Solubilization and partial purification of the high-affinity gonadoliberin receptor from the bovine pituitary gland.
The high-affinity gonadoliberin (GnRH) receptor contained in a membrane preparation from frozen bovine anterior pituitary glands has been solubilized in Triton X-100 and the binding properties of the solubilized product have been examined. The radioreceptor-binding assay, using the GnRH agonist [D-Ser(t-Bu)6] des-Gly10GnRH N-ethylamide (GnRH-A) as radioligand, demonstrated that the kinetics of association and dissociation, the binding constants, as well as the specificity of receptor were not altered in the solubilized receptor preparations. Affinity chromatography on a concanavalin A-Sepharose column, with elution of adsorbed material using a solution of alpha-methyl-D-mannoside, allowed a 33-fold purification of the receptor. The Ka of the receptor thus purified was of the same order as that of the starting material, although slightly higher values were found. Only about one-half of the total receptor activity applied to the column was retained in spite of several recyclings. The other half was found in the nonadsorbed fraction. It is postulated that the detergent-solubilized fraction contains two forms of the GnRH receptor. The nonadsorbed fraction probably contains a partially or totally deglycosylated form. It is possible that the detergent-solubilization process somewhat alters the physicochemical properties of a part of the GnRH receptor molecules. Electrophoretic analysis of the purified receptor preparations, with a subsequent GnRH-binding assay, suggests that the apparent molecular mass of the high-affinity GnRH receptor, or of its monomeric form, is approximately 60,000 Da.
D000818:Animals; D001667:Binding, Competitive; D002417:Cattle; D007987:Gonadotropin-Releasing Hormone / Q000378:metabolism*; D007700:Kinetics; D008970:Molecular Weight; D010903:Pituitary Gland, Anterior / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000302:isolation & purification / Q000378:metabolism*; D011966:Receptors, LHRH; D012995:Solubility
D011956:Receptors, Cell Surface; D011966:Receptors, LHRH; D007987:Gonadotropin-Releasing Hormone