Purification of TRH receptor and its mRNA

将猪和大鼠垂体前叶的粗质膜在含有1%毛地黄皂苷的40 mM Tris HCl缓冲液中匀浆，0 ℃孵育30 min，100，000 × g离心60 min。所得上清液与（3 h-MetHis）TRH在0 ℃孵育3 h，通过快速凝胶过滤分离结合的标记TRH。1%毛地黄皂苷产生40%的溶解的原始TRH受体密度。在使用Sorbax柱的HPLC上观察到（3 h）TRH标记的TRH受体的峰对应于（14 C）肌球蛋白的峰，数据表明猪垂体前叶中TRH受体的分子量约为200 × K。溶解的猪TRH受体被麦胚凝集素柱吸附，吸附的受体被0.3M N-乙酰氨基葡萄糖洗脱。这些数据表明TRH受体具有N-乙酰基-D-葡糖胺基残基。观察到溶解的猪TRH受体的Kd=30 nM，Bmax=62 fmol/mg蛋白质。用溶解的猪TRH受体和（~ 3 H）TRH孵育18例桥本甲状腺炎患者血清，以研究抗TRH受体抗体的可能存在。然而，这些血清没有改变TRH结合到猪垂体前叶。将溶解的TRH受体施加到与TRH偶联的AH-sephadex 4 B柱上，并用未标记的TRH洗脱。这种TRH亲和层析系统可以产生约200倍浓度的猪TRH受体。进一步的研究正在进行中，以获得1,000倍浓缩的TRH受体，并确定对应于猪TRH受体氨基酸序列的寡核苷酸探针。

The crude plasma membrane of porcine and rat anterior pituitaries was homogenized in 40mM Tris HCl buffer containing 1% digitonin, incubated at 0 C for 30 min, and centrifuged at 100,000xg for 60 min. The resulting supernatant was incubated with (3h-MetHis)TRH at 0 C for 3 h, and the bound labeled TRH was separated by means of a rapid gel filtration. One% digitonin yielded to 40% solubilization of the original TRH receptor density. The peak of (3h) TRH-labeled TRH receptor was observed to correspond to that of (14C)myosin on a HPLC using Sorbax column, the data suggesting that a molecular weight of TRH receptor in the porcine anterior pituitary is approximately 200xK. The solubilized porcine TRH receptor was adsorbed by a wheat germ agglutinin column, and the adsorbed receptor eluted by 0.3M N-acetyl glucosamine. The data indicate that TRH receptor possesses N-acetyl-D-glucosaminyl residues. The solubilized porcine TRH receptor was observed to be Kd=30 nM and Bmax=62 fmol/mg protein. The sera from 18 patients with Hashimoto's thyroiditis were incubated with the solubilized porcine TRH receptor and (3H)TRH, to investigate the possible existence of an anti-TRH receptor antibody. However, those sera did not change TRH bound to the porcine anterior pituitary. The solubilized TRH receptor was applied to a AH-sephadex 4B coulumn coupled with TRH, and eluted by unlabeled TRH. This TRH-affinity chromatography system could result in an approximately 200- fold concentration of porcine TRH receptor. Further studies are in progress to obtain the 1,000-fold concentrated TRH receptor and determine an oligonucleotide probe corresponding to amino-acid sequence of porcine TRH receptor.