Herington A C, Elson D, Ymer S
J Recept Res. 1981;2(2):203-20. doi: 10.3109/10799898109039261.
Soluble receptors that bind human growth hormone have been prepared by incubation of liver membranes form pregnant female rabbits in 1 mM Tris buffer (ph 7.5 or 9.0) at 4 degrees C. Up to 29% of the growth hormone binding sites could be solubilized within 48 hours. The kinetics of binding of human growth hormone to the soluble receptor, the hormonal specificity and the binding parameters calculated by Scatchard analysis (Ka 2.2 x 10(9) M-1, capacity 409 fmole/mg) were essentially unchanged compared with those for the parent membrane-associated (particulate) receptor. Gel filtration on Ultrogel AcA22 indicated that the major binding peak eluted at a molecular weight of 300,000 daltons. Specificity studies showed that the soluble binding sites had a moderately high affinity for ovine prolactin (Ka integral of 1 x 10(8) M-1), but negligible affinity for insulin. Although aqueous extraction gives a lower yield of binding sites for human growth hormone than detergent extraction, it nevertheless avoids some of the problems associated with use of detergents and should facilitate the subsequent purification of the receptor in a relatively unaltered state. It may also have applicability for solubilization of other hormone receptor systems.
通过将来自怀孕雌兔的肝膜在4℃下于1 mM Tris缓冲液(pH 7.5或9.0)中孵育,制备了与人生长激素结合的可溶性受体。在48小时内,高达29%的生长激素结合位点可被溶解。与亲本膜相关(颗粒状)受体相比,人生长激素与可溶性受体结合的动力学、激素特异性以及通过Scatchard分析计算的结合参数(Ka 2.2×10⁹ M⁻¹,容量409 fmole/mg)基本未变。在Ultrogel AcA22上进行凝胶过滤表明,主要结合峰在分子量300,000道尔顿处洗脱。特异性研究表明,可溶性结合位点对绵羊催乳素具有中等高度的亲和力(Ka积分1×10⁸ M⁻¹),但对胰岛素的亲和力可忽略不计。尽管水相提取得到的人生长激素结合位点产量低于去污剂提取,但它仍避免了一些与使用去污剂相关的问题,并且应有助于以相对未改变的状态随后纯化受体。它也可能适用于其他激素受体系统的溶解。