Bramley T A, Stirling D, Swanston I A, Menzies G S, Baird D T
J Endocrinol. 1987 May;113(2):305-15. doi: 10.1677/joe.0.1130305.
The specific binding of 125I-labelled human chorionic gonadotrophin (hCG), human low-density lipoprotein (hLDL), human FSH (hFSH) and human prolactin (hPRL) to homogenates of human corpus luteum tissue was measured. Specific binding of 125I-labelled hCG was dependent on the temperature and duration of incubation, was inhibited by divalent metal ions or chelating agents, and increased linearly with homogenate concentration. Recovery of bound hormone was more effective using Millipore filtration or polyethylene glycol precipitation compared with centrifugation alone. Binding of 125I-labelled hCG was inhibited specifically by low levels of hCG and human LH (hLH) but not by ovine LH or bovine LH. Incubation of human luteal tissue with ice-cold citrate buffer (pH 3) released more than 90% of specifically bound 125I-labelled hCG within 5 min. This treatment inactivated LH receptors, but did not affect the immunoactivity of hLH released, enabling the measurement of released hormone by radioimmunoassay. Scatchard plots of binding of 125I-labelled LDL to human corpus luteum demonstrated a single class of binding sites. Binding was saturable, increased linearly with increasing concentration of homogenate, and was displaceable by low concentrations of unlabelled LDL. Binding of 125I-labelled hPRL to human luteal homogenates was increased by Mg2+ and was specific for lactogenic hormones (human prolactin, human growth hormone and ovine prolactin). Binding of 125I-labelled hFSH was not dependent on divalent metal ion concentration (in marked contrast to hFSH binding to immature pig granulosa cell receptors) and was displaced by hFSH preparations but not by hPRL, ovine LH or hCG at 1 microgram/ml. These results establish optimal conditions and hormone specificities for the measurement of human luteal gonadotrophin and LDL receptors, and methods for the estimation of hLH/hCG endogenously bound to human corpus luteum tissue.
测定了¹²⁵I标记的人绒毛膜促性腺激素（hCG）、人低密度脂蛋白（hLDL）、人促卵泡激素（hFSH）和人催乳素（hPRL）与人黄体组织匀浆的特异性结合。¹²⁵I标记的hCG的特异性结合取决于孵育温度和时间，受二价金属离子或螯合剂抑制，并随匀浆浓度呈线性增加。与单独离心相比，使用密理博过滤或聚乙二醇沉淀回收结合激素的效果更好。¹²⁵I标记的hCG的结合被低水平的hCG和人促黄体生成素（hLH）特异性抑制，但不被羊促黄体生成素或牛促黄体生成素抑制。用人黄体组织与冰冷的柠檬酸盐缓冲液（pH 3）孵育，5分钟内释放出超过90%的特异性结合的¹²⁵I标记的hCG。这种处理使促黄体生成素受体失活，但不影响释放的hLH的免疫活性，从而能够通过放射免疫测定法测量释放的激素。¹²⁵I标记的低密度脂蛋白与人黄体结合的Scatchard图显示为单一类别的结合位点。结合是可饱和的，随匀浆浓度增加呈线性增加，并且可被低浓度的未标记低密度脂蛋白取代。¹²⁵I标记的hPRL与人黄体匀浆的结合被Mg²⁺增强，并且对催乳激素（人催乳素、人生长激素和羊催乳素）具有特异性。¹²⁵I标记的hFSH的结合不依赖于二价金属离子浓度（这与hFSH与未成熟猪颗粒细胞受体的结合形成鲜明对比），并且被hFSH制剂取代，但不被1微克/毫升的hPRL、羊促黄体生成素或hCG取代。这些结果确定了测量人黄体促性腺激素和低密度脂蛋白受体的最佳条件和激素特异性，以及估计内源性与人黄体组织结合的hLH/hCG的方法。
