Solubilization and partial characterization of angiotensin II receptors from rat brain.
Solubilization and partial characterization of angiotensin II receptors from rat brain.
复制标题
大鼠脑血管紧张素 II 受体的溶解和部分表征。
DOI：
10.1111/j.1471-4159.1991.tb03801.x
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发表时间：
1991
影响因子：
4.7
通讯作者：
Harding,JW
中科院分区：
文献类型：
--
作者：
Siemens,IR;Swanson,GN;Fluharty,SJ;Harding,JW
使用合成洗涤剂 3[(3-胆酰胺丙基)二甲基铵)]-1-丙烷磺酸盐溶解大鼠脑血管紧张素 II (Ang II) 受体，产率为 30-40%。使用高亲和力拮抗剂 I25I-Sar1、lle8-Ang II 进行的动力学分析表明，溶解的受体表现出与完整脑膜内存在的受体相同的特性。此外，在溶解的或完整的膜中竞争125I-Sarl,IIe8-Ang II标记的结合位点的一系列激动剂和拮抗剂各自的pIC50值之间存在正相关（r -0.99）。此外，用同源双功能交联剂二琥珀酰亚胺基辛二酸酯将 125 I-Ang II 共价标记到溶解的受体上，然后进行凝胶过滤，显示出一个主要和一个次要结合峰，表观分子量分别为 64,000 和 115,000。通过 I25l-Ang II 与溶解脑膜的共价交联，然后进行十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分析，还鉴定了两种分子量相当的结合蛋白（即 112,000 和 60,000）。相比之下，当在凝胶过滤之前用拮抗剂 125I-Sar1.IIe8-Ang II 标记溶解的膜时，仅观察到分子量较小的结合蛋白，并且拮抗剂标记位点的色谱聚焦仅显示一个等角点为 6.2 的峰。这些结合位点的成功溶解应有助于继续研究大脑中的 Ang II 受体。
Rat brain angiotensin II (Ang II) receptors were solubilized with a yield of 30–40% using the synthetic detergent 3[(3‐cholamidopropyl)dimethylammonio)]‐1 ‐propane‐sulfonate. Kinetic analysis employing the high‐affinity antagonistI25I‐Sar1, lle8‐Ang II indicated that the solubilized receptors exhibited the same properties as receptors present within intact brain membranes. Furthermore, there was a positive correlation (r −0.99) between the respective pIC50values of a series of agonist and antagonists competing for125I‐Sarl,IIe8‐Ang II labeled binding sites in either solubilized or intact membranes. Moreover, covalent labeling of125I‐ Ang II to solubilized receptors with the homo‐bifunctional cross‐linker disuccinimidyl suberate, followed by gel filtration, revealed one major and one minor binding peak with apparent molecular weights of 64,000 and 115,000, respectively. Two binding proteins of comparable molecular weights (i.e., 112,000 and 60,000) were also identified by covalent cross‐linking ofI25l‐Ang II to solubilized brain membranes followed by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis analysis. In contrast, only the smaller molecular mass binding protein was observed when solubilized membranes were labeled with the antagonist125I‐Sar1.IIe8‐Ang II prior to gel filtration, and chromatofocusing of antagonist labeled sites revealed only one peak with an isodectric point of 6.2. The successful solubilization of these binding sites should facilitate continued investigation of Ang II receptors in the brain.