Rogers T B
J Biol Chem. 1984 Jul 10;259(13):8106-14.
High affinity receptors for angiotensin II have been identified on purified cardiac sarcolemmal membranes. Equilibrium binding studies were performed with 125I-labeled angiotensin II and purified sarcolemmal vesicles from calf ventricle. The curvilinear Scatchard plots were evaluated by nonlinear regression analysis using a two-site model which identified a high affinity site Kd1 = 1.08 +/- 0.3 nM and N1 = 52 +/- 10 fmol/mg of protein and a low affinity site Kd2 = 52 +/- 16 nM and N2 = 988 +/- 170 fmol/mg of protein. Monovalent and divalent cations inhibited the binding of 125I-angiotensin II by 50%. The affinity of angiotensin II analogs for the receptor was determined using competitive binding assays; sarcosine, leucine-angiotensin II (Sar,Leu-angiotensin II), Kd = 0.53 nM; angiotensin II, Kd = 2.5 nM; des-aspartic acid-angiotensin II, Kd = 4.81 nM; angiotensin I, Kd = 77.6 nM. There is a positive correlation between potency in inducing positive inotropic response in myocardial preparations reported by others and potency for the hormone receptor observed in the binding assays. Pseudo-Hill plots of the binding data showed that agonists display biphasic binding with Hill numbers around 0.65 while antagonists recognized a single class of high affinity receptors with Hill numbers close to unity. These data were confirmed using 125I-Sar,Leu-angiotensin II in equilibrium binding studies which showed that this antagonist bound to a single class of receptor sites; Kd = 0.42 +/- 0.04 nM and N = 1050 +/- 110 fmol/mg of protein. Competition-binding experiments with this 125I-peptide yielded monophasic curves with Hill numbers close to unity for both agonists and antagonists. Membrane-bound 125I-angiotensin II was covalently linked to its receptor by the use of bifunctional cross-linking reagents such as dithiobis(succinimidyl propionate) and bis[2-(succinimidooxycarbonyloxy)ethyl]sulfone. Analysis of the membranes showed the labeling of a component with an apparent Mr = 116,000. The affinity labeled species showed characteristics expected of a functional component of the high affinity receptor. The affinity labeling of this membrane component was inhibited by nanomolar angiotensin II or Sar,Leu-angiotensin II. Together these data indicate that high affinity receptors exist for angiotensin II that most likely mediate the positive inotropic effects of this hormone on myocardial cells.(ABSTRACT TRUNCATED AT 400 WORDS)
在纯化的心肌肌膜上已鉴定出血管紧张素 II 的高亲和力受体。使用 125I 标记的血管紧张素 II 和从小牛心室纯化的肌膜囊泡进行平衡结合研究。通过使用双位点模型的非线性回归分析评估曲线型 Scatchard 图，该模型确定了一个高亲和力位点 Kd1 = 1.08 +/- 0.3 nM 和 N1 = 52 +/- 10 fmol/mg 蛋白质，以及一个低亲和力位点 Kd2 = 52 +/- 16 nM 和 N2 = 988 +/- 170 fmol/mg 蛋白质。单价和二价阳离子抑制 125I - 血管紧张素 II 的结合达 50%。使用竞争性结合测定法确定血管紧张素 II 类似物对受体的亲和力；肌氨酸、亮氨酸 - 血管紧张素 II（Sar,Leu - 血管紧张素 II），Kd = 0.53 nM；血管紧张素 II，Kd = 2.5 nM；去天冬氨酸 - 血管紧张素 II，Kd = 4.81 nM；血管紧张素 I，Kd = 77.6 nM。其他人报道的在心肌制剂中诱导正性肌力反应的效力与结合测定中观察到的激素受体效力之间存在正相关。结合数据的伪希尔图表明，激动剂表现出双相结合，希尔系数约为 0.65，而拮抗剂识别一类单一的高亲和力受体，希尔系数接近 1。在平衡结合研究中使用 125I - Sar,Leu - 血管紧张素 II 证实了这些数据，该研究表明这种拮抗剂与一类单一的受体位点结合；Kd = 0.42 +/- 0.04 nM 和 N = 1050 +/- 110 fmol/mg 蛋白质。用这种 125I - 肽进行的竞争结合实验对激动剂和拮抗剂均产生希尔系数接近 1 的单相曲线。通过使用双功能交联试剂如二硫代双（琥珀酰亚胺丙酸酯）和双[2 - （琥珀酰亚胺氧基羰基氧基）乙基]砜，将膜结合的 125I - 血管紧张素 II 与其受体共价连接。对膜的分析显示标记了一个表观 Mr = 116,000 的成分。亲和标记的物种表现出高亲和力受体功能成分所预期的特征。这种膜成分的亲和标记被纳摩尔浓度的血管紧张素 II 或 Sar,Leu - 血管紧张素 II 抑制。这些数据共同表明存在血管紧张素 II 的高亲和力受体，其很可能介导该激素对心肌细胞的正性肌力作用。（摘要截短至 400 字）
