Sairam M R, Yarney T A, Bhargavi G N, Sanford L M
Reproduction Research Laboratory, Clinical Research Institute of Montreal, Quebec, Canada.
J Biol Chem. 1988 Mar 15;263(8):3706-12.
A membrane preparation from the testis of maturing Dorset-Leicester-Suffolk sheep, capable of discriminating pituitary LH (lutropin) from placental gonadotropins human choriogonadotropin (hCG) and equine choriogonadotropin is described. Maximum binding of 125I-oLH (ovine lutropin) to the testicular receptors occurred at 4 degrees C in a rapid manner, attaining equilibrium in 12-16 h. Under such optimal conditions, only unlabeled ovine LH or the structurally identical bovine LH effectively competed for receptor occupation. Other highly purified pituitary LH preparations from rat and human pituitaries were weakly (4-10%) active in displacement assays. Purified hCG or equine choriogonadotropin, which were highly potent in rat testicular LH receptor assays, could not compete with 125I-oLH for binding to the sheep LH receptor at 4 degrees C. Thus, the sheep testicular LH receptor was highly specific in recognizing pituitary LH conformation. The presence of an ovine/bovine LH alpha- or beta-subunit in recombinants with hCG subunit counterparts was required to generate an effective conformation capable of receptor recognition. Chemically deglycosylated hCG, containing 75% less carbohydrate and which showed greater binding to other LH receptors, failed to recognize sheep LH receptor, suggesting that excess carbohydrate in hCG was not a factor in hindering binding of the native placental hormone. Scatchard analysis using 125I-hCG/125I-oLH revealed that there were separate sites with similar affinities but vastly different capacities. The hCG binding sites, which could also be effectively occupied by oLH, were less than 10% of oLH binding sites. Thus, the Dorset-Leicester-Suffolk sheep testicular receptor provides an important and unique in vitro test system to distinguish pituitary LH from placental LH-like hormones. We infer that temperature-dependent conformational restrictions of the sheep testicular LH receptor are involved in recognizing differences in these highly similar and structurally homologous hormones.
本文描述了一种从成熟的多塞特-莱斯特-萨福克绵羊睾丸制备的膜制剂，该制剂能够区分垂体促黄体生成素（促性腺激素）与胎盘促性腺激素——人绒毛膜促性腺激素（hCG）和马绒毛膜促性腺激素。125I-oLH（绵羊促性腺激素）与睾丸受体的最大结合在4℃时迅速发生，12-16小时达到平衡。在这种最佳条件下，只有未标记的绵羊促黄体生成素或结构相同的牛促黄体生成素能有效竞争受体占据。来自大鼠和人垂体的其他高度纯化的垂体促黄体生成素制剂在置换试验中的活性较弱（4-10%）。在大鼠睾丸促黄体生成素受体试验中效力很强的纯化hCG或马绒毛膜促性腺激素，在4℃时不能与125I-oLH竞争结合绵羊促黄体生成素受体。因此，绵羊睾丸促黄体生成素受体在识别垂体促黄体生成素构象方面具有高度特异性。与hCG亚基对应物重组时，需要存在绵羊/牛促黄体生成素α或β亚基，以产生能够被受体识别的有效构象。化学去糖基化的hCG，碳水化合物含量减少75%，与其他促黄体生成素受体的结合更强，但不能识别绵羊促黄体生成素受体，这表明hCG中过量的碳水化合物不是阻碍天然胎盘激素结合的因素。使用125I-hCG/125I-oLH进行的Scatchard分析表明，存在具有相似亲和力但容量差异很大的不同位点。hCG结合位点也可被oLH有效占据，其数量不到oLH结合位点的10%。因此，多塞特-莱斯特-萨福克绵羊睾丸受体提供了一个重要且独特的体外测试系统，用于区分垂体促黄体生成素与胎盘类促黄体生成素激素。我们推断，绵羊睾丸促黄体生成素受体的温度依赖性构象限制参与了识别这些高度相似且结构同源的激素之间的差异。
