Flanagan C A, Tetaz T, Smith A I, Millar R P
Department of Chemical Pathology, University of Cape Town Medical School, Observatory, South Africa.
Biomed Chromatogr. 1996 Mar-Apr;10(2):83-8. doi: 10.1002/(SICI)1099-0801(199603)10:2<83::AID-BMC563>3.0.CO;2-Z.
Gonadotropin-releasing hormone (GnRH) is the primary regulator of mammalian reproduction. It stimulates the release of luteinizing hormone and follicle stimulating hormone via receptors on the cell membranes of pituitary gonadotrope cells. This paper describes the development of a protocol for purification of GnRH binding proteins from sheep pituitary membranes. Membranes were best solubilized using a zwitterionic detergent. Solubilized membranes were applied to an affinity column prepared with a GnRH analogue. The most effective analogue was the agonist [D-Lys6,Pro9-NHEt]-GnRH. The column was washed with a gradient of sodium chloride up to 0.4 M and GnRH binding activity was eluted from the column using the acidic buffer. Eluted fractions bound labelled GnRH agonist after neutralization of the buffer. Sodium dodecyl sulphate polyacrylamide gel electrophoresis analysis revealed a major protein band with a relative molecular weight of 67 kD. Amino acid sequence analysis showed that the protein is different from the cloned GnRH receptor, but homologous with a similar protein recently purified from bovine pituitary. This protein may have a function which is modulated by binding of GnRH, GnRH fragments or GnRH-related peptides.
促性腺激素释放激素（GnRH）是哺乳动物生殖的主要调节因子。它通过垂体促性腺激素细胞细胞膜上的受体刺激黄体生成素和卵泡刺激素的释放。本文描述了一种从绵羊垂体膜中纯化GnRH结合蛋白的方法的开发。使用两性离子去污剂能最好地溶解膜。将溶解的膜应用于用GnRH类似物制备的亲和柱。最有效的类似物是激动剂[D-Lys6,Pro9-NHEt]-GnRH。用高达0.4 M的氯化钠梯度洗涤柱子，并用酸性缓冲液从柱上洗脱GnRH结合活性。缓冲液中和后，洗脱的级分结合标记的GnRH激动剂。十二烷基硫酸钠聚丙烯酰胺凝胶电泳分析显示一条主要蛋白带，相对分子质量为67 kD。氨基酸序列分析表明该蛋白与克隆的GnRH受体不同，但与最近从牛垂体中纯化的类似蛋白同源。这种蛋白可能具有一种功能，该功能可通过GnRH、GnRH片段或GnRH相关肽的结合来调节。
