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Purification of TRH receptor and its mRNA

TRH受体及其mRNA的纯化

基本信息

批准号：
60570516
负责人：
MORI Masatomo
金额：
$ 1.09万
依托单位：
GUNMA UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1985
资助国家：
日本
起止时间：
1985 至 1987
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-60570516/
关键词：
TRH TRH receptor mRNA TRHreceptor

项目摘要

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.

将猪和大鼠垂体前叶的粗质膜在含1%洋地黄素的40 mM Tris HCl缓冲液中匀浆，0℃孵育30分钟，100,000×g离心60分钟。将得到的上清液与(3h-MetHis)TRH在0℃孵育3h，通过快速凝胶过滤分离结合的标记TRH。1%洋地黄素使原TRH受体密度增加40%。用Sorbax色谱柱测定~(3)H-TRH标记的TRH受体的峰与~(14)C-肌球蛋白的峰相对应，提示猪垂体前叶TRH受体的相对分子质量约为200×K。用麦芽凝集素柱吸附溶解的猪TRH受体，用0.3M N-乙酰氨基葡萄糖洗脱。结果表明，TRH受体含有N-乙酰-D-氨基葡萄糖残基。溶解的猪TRH受体的Kd=30 nM，Bmax=62fmol/mg蛋白。用溶解的猪TRH受体和(~3H)TRH与18例桥本甲状腺炎患者血清孵育，以探讨抗TRH受体抗体的可能存在。然而，这些血清不改变与猪垂体前叶结合的TRH。将溶解的TRH受体应用于与TRH偶联的AH-Sephadex 4B柱上，并用未标记的TRH洗脱。这个TRH亲和层析系统可以得到大约200倍的猪TRH受体浓度。进一步的研究正在进行中，以获得1000倍浓缩的TRH受体，并确定与猪TRH受体的氨基酸序列相对应的寡核苷酸探针。