Covalent crosslinking of angiotensin II to its binding sites in rat adrenal membranes.
Covalent crosslinking of angiotensin II to its binding sites in rat adrenal membranes.
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血管紧张素 II 与大鼠肾上腺膜结合位点的共价交联。
DOI：
10.1073/pnas.79.12.3739
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发表时间：
1982
影响因子：
11.1
通讯作者：
Jamieson,JD
中科院分区：
文献类型：
--
作者：
Paglin,S;Jamieson,JD
为了鉴定肾上腺皮质血管紧张素II （AII）受体的分子成分，我们使用化学交联剂二琥珀酰亚酸将125i标记的AII （125I-AII）与其结合位点共价连接。大鼠肾上腺组织的膜组分用125I-AII孵育，洗涤，并用二琥珀酰亚基处理。在还原条件下进行NaDodSO4/聚丙烯酰胺凝胶电泳，然后进行放射自显像，结果显示平均Mr为11.6万的主要多肽被125I-AII共价标记。Mr值为54,000和45,000的蛋白质也被轻轻标记。Mr 116,000种的标记是特异性的，因为它可以通过预先用[Sar1，Leu8]AII或未标记的AII孵育膜而消除。预先用[Sar1，Leu8]AII孵育对较小蛋白的标记影响较小。在多种蛋白酶抑制剂存在的情况下进行交联的实验表明，Mr 116000种不是参与AII降解的酶。非还原条件下的凝胶电泳表明，该组分不是来自较大的二硫化物连接实体。我们认为Mr 116,000部分是AII受体系统的一部分。Mr 54000和Mr 45000多肽与AII受体的关系，如果有的话，仍然是未知的。
To identify the molecular components of the angiotensin II (AII) receptor in the adrenal cortex, we have covalently linked 125I-labeled AII (125I-AII) to its binding sites by using the chemical crosslinker disuccinimidyl suberate. Membrane fractions from rat adrenal tissue were incubated with 125I-AII, washed, and treated with disuccinimidyl suberate. NaDodSO4/polyacrylamide gel electrophoresis carried out under reducing conditions followed by autoradiography showed that a major polypeptide(s) with an average Mr of 116,000 was covalently tagged with 125I-AII. Proteins with Mr of 54,000 and 45,000 were also lightly labeled. Labeling of the Mr 116,000 species was specific in that it could be abolished by prior incubation of the membranes with [Sar1,Leu8]AII or unlabeled AII. Labeling of the smaller proteins was less affected by prior incubation with [Sar1,Leu8]AII. Experiments in which crosslinking was carried out in the presence of a variety of protease inhibitors indicated that the Mr 116,000 species is not an enzyme involved in AII degradation. Gel electrophoresis under nonreducing conditions showed that this component is not derived from larger disulfide-linked entities. We suggest that the Mr 116,000 moiety is a part of the receptor system for AII. The relationship, if any, of the Mr 54,000 and 45,000 peptides to the AII receptor remains unknown.