Solubilization and characterization of an angiotensin II binding protein from liver.
Solubilization and characterization of an angiotensin II binding protein from liver.
复制标题
肝脏血管紧张素 II 结合蛋白的溶解和表征。
DOI：
10.1111/j.1432-1033.1983.tb07702.x
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发表时间：
1983
期刊：
影响因子：
--
通讯作者：
Soffer,RL
中科院分区：
文献类型：
--
作者：
Sen,I;Jim,KF;Soffer,RL
与血管紧张素II的高亲和力的结合位点溶解从肝细胞膜处理毛地黄皂苷。放射性碘标记的血管紧张素II的结合测定凝胶过滤和独立的技术，利用活性炭吸附结合配体的失败。使用硫酸铵分级分离，然后进行凝胶过滤，部分纯化结合蛋白，在蛋白酶抑制剂的存在下，分离的结合蛋白制剂不会催化血管紧张素II的降解。与膜以及溶解制剂的结合是特异性的和饱和的。膜显示出单组高亲和力结合位点，aKd为0.5 nM。溶解的制剂也显示存在单一类别的高亲和力结合位点（Kd= 10.5 nM）。使用血管紧张素I以及血管紧张素II的各种片段、激动剂和拮抗剂的置换研究揭示了与完整膜相似的结构活性特征。血管紧张素II从可溶性大分子复合物中的解离缓慢，但在非生理pH值或存在4.5 M尿素或1%十二烷基硫酸钠时增强。放射性碘标记的血管紧张素II与一个单一的，特定的大分子成分的共价结合，通过处理与二琥珀酰亚胺辛二酸酯。通过聚丙烯酰胺凝胶电泳，这种还原的变性放射性蛋白的表观分子量估计为约68000。
Binding sites with high affinity for angiotensin II were solubilized from hepatic membranes by treatment with digitonin. Binding of radioiodinated angiotensin II was assayed by gel filtration and independently by a technique exploiting the failure of activated charcoal to adsorb the bound ligand. The binding protein was partially purified using ammonium sulfate fractionation followed by gel filtration, and in the presence of protease inhibitors, the isolated binding protein preparation did not catalyze degradation of the angiotensin II.Binding to the membranes as well as to the solubilized preparation was specific and saturable. The membranes exhibited a single set of high‐affinity binding sites with aKdof 0.5 nM. The solubilized preparation, also showed the presence of a single class of high‐affinity binding sites (Kd= 10.5 nM). Displacement studies using angiotensin I as well as various fragments, agonists and antagonists of angiotensin II disclosed a structure‐activity profile similar to that found with intact membranes. Dissociation of angiotensin II from the soluble macromolecular complex was slow but was enhanced at non‐physiological pH values or in the presence of 4.5 M urea, or 1% sodium dodecyl sulfate. Covalent binding of the radioiodinated angiotensin II to a single, specific macromolecular component was achieved by treatment with disuccinimidyl suberate. The apparent molecular weight of this reduced, denatured radioactive protein was estimated at about 68000 by polyacrylamide gel electrophoresis.