A novel angiotensin analog with subnanomolar affinity for angiotensin-converting enzyme

作     者：Krebs, LT Hanesworth, JM Sardinia, MF Speth, RC Wright, JW Harding, JW 

作者机构：Washington State Univ Dept VCAPP Pullman WA 99164 USA 

出 版 物：《JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS》 (药理学与实验治疗学杂志)

年 卷 期：2000年第293卷第1期

页      面：260-267页

核心收录：

学科分类：1007[医学-药学(可授医学、理学学位)] 10[医学] 

主　　题：血管紧张素Ⅰ/类似物和衍生物 血管紧张素Ⅰ/药理学 血管紧张素转换酶抑制药/药理学 血管紧张素类/药理学 放射自显影术 结合 竞争性/药物作用 缓激肽/药理学 化学 物理 交联试剂 电泳 聚丙烯酰氨凝胶 指示剂和试剂 碘放射性同位素/诊断应用 肺/药物作用 肺/代谢 肽基二肽酶A/血液 肽基二肽酶A/代谢 蛋白质结合 动物 豚鼠 男(雄)性 

摘      要：This study demonstrates that a novel angiotensin I analog, angiotensinogen 3-11(Lys(11)), possesses a high affinity for angiotensin-converting enzyme (ACE), which is substantially greater than the endogenous substrates. This assessment is based on data derived from a variety of techniques. First, the binding characteristics of I-125-angiotensinogen 3-11(Lys(11)) were examined. Equilibrium saturation isotherms utilizing guinea pig lung membranes revealed that I-125-angiotensinogen 3-11(Lys(11)) bound a single high-affinity site in the presence of EDTA exhibiting a K sigma of 0.15 +/- 0.02 nM with a B-max = 4295 +/- 535 fmol/mg of protein. Competition studies revealed the following rank order of binding affinity: I-125-angiotensinogen 3-11(Lys(11)) bradykinin angiotensin I. Next, SDS-polyacrylamide gel electrophoresis analysis revealed that chemically cross-linked I-125-angiotensinogen 3-11(Lys(11)) specifically bound a protein of M-r 173,000 that had the same molecular weight as ACE. Utilizing in vitro autoradiography, the binding distributions of I-125-angiolensinogen 3-11(Lys(11)) and the ACE inhibitor, I-125-351A, were also compared. These experiments demonstrated that the binding distributions of I-125-angiotensinogen 3-11(Lys(11)) and I-125-351A are identical in the guinea pig lung and testes. Finally, the purification of ACE from guinea pig serum was monitored with I-125-angiotensinogen 3-11(Lys(11)) and I-125-351A binding. These results demonstrated that the binding site for I-125-angiotensinogen 3-11(Lys(11)) and I-125-351A copurified. These experiments indicate that the novel angiotensin I analog, I-125-angiotensinogen 3-11(Lys(11)) binds to ACE and suggest that there are critical binding sites outside the catalytic domains of ACE that determine binding specificity and affinity.