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

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.

将猪和大鼠前垂体的粗质膜在含有1％digitonin的40mm Tris HCl缓冲液中匀浆，在0 C下孵育30分钟，并以100,000xg离心60分钟。将所得的上清液与（3H-METHIS）TRH在0 C下孵育3小时，并通过快速的凝胶过滤将被标记的TRH孵育。 1％的digitonin屈服于原始TRH受体密度的40％溶解化。观察到（3H）TRH标记的TRH受体的峰使用Sorbax柱上的HPLC上的（14C）肌球蛋白的峰对应于（14C）肌球蛋白的峰，这些数据表明猪前垂体中TRH受体的分子量大约为200xK。溶解的猪TRH受体被小麦胚芽凝集素柱吸附，并通过0.3m n-乙酰葡萄糖胺洗脱了吸附的受体。数据表明TRH受体具有N-乙酰基-D-葡萄糖胺基残基。观察到溶解的猪TRH受体为KD = 30 nm，BMAX = 62 FMOL/mg蛋白。将18例桥本甲状腺炎患者的血清与溶解的猪TRH受体和（3H）TRH孵育，以研究抗TRH受体抗体的可能存在。但是，这些血清没有改变与猪前垂体结合的TRH。将溶解的TRH受体应用于与TRH结合的AH-Sephadex 4B，并被未标记的TRH洗脱。这种TRH亲和力色谱系统可能导致大约200倍的猪TRH受体浓度。进一步的研究正在进行中，以获得1,000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。