Michel B, Nirina L B, Grima M, Ingert C, Coquard C, Barthelmebs M, Imbs J L
Institut de pharmacologie, faculté de médecine, université Louis-Pasteur, Strasbourg.
Arch Mal Coeur Vaiss. 1998 Aug;91(8):1013-9.
Somatic angiotensin-converting enzyme (ACE) is a protein which contains two similar domains (N and C), each possessing a functional active site. The relationship between ACE, its natural substrates and oxygen free radicals is starting to be explored. On one hand, superoxide anions production is induced by angiotensin II and on the other hand, activated polynuclear neutrophils, through free radicals generation, alter endothelial ACE activity. In this study, we examined the impact of hydroxyl radicals (.OH) on purified ACE. .OH were produced using a generator: 2,2'-azo-bis 2-amidinopropane (GRH) provided by Lara-Spiral (Fr). GRH (3 mM), in a time-dependent fashion, inhibited ACE activity. When ACE was co-incubated for 4 h with GRH, its activity decreased by 70%. Addition of dimethylthiourea (DMTU: 0.03 to 1 mM) or mannitol + methionine (20/10 mM), two sets of .OH scavengers, produced a dose-dependent protection on ACE activity. To examine whether oxidation of thiol groups in the ACE molecule could be involved in the action of GRH, the effects of thiol reducing agents: mercaptoethanol and dithiotreitol (DTT) were investigated. These compounds produced a dose-dependent and significant protection; with 100% protection at 0.2 and 0.3 mM for mercaptoethanol and at 0.1 mM for DTT. The hydrolysis of two natural and domain-specific substrates were also explored. The hydrolysis of angiotensin I preferentially cleaved by the C domain was significantly (p < 0.01) inhibited by 57, 58 and 69% in contact with 0.3, 1 and 3 mM GRH [in nmol angio II formed/min/nmol of ACE, n = 4; 35.9 +/- 0.6 (control), 15.5 +/- 2.8 (GRH : 0.3 mM), 15.1 +/- 0.5 (1), 10.9 +/- 0.6 (3)]. The hydrolysis of the hemoregulatory peptide (hp), preferential substrate for the N domain was not affected by GRH at 0.3 mM and inhibited by 28% (not significant) by 1 mM GRH [in nmol ph hydrolized/min/nmol ACE, n = 4; 12.6 +/- 1.9 (control), 14.9 (GRH : 0.3 mM), 8.3 +/- 4.0 (1). These results demonstrated that .OH affect ACE activity and could suggest a privileged impact of GRH on the C domain. The precise sites of action of .OH remain unknown. The Cys residues near the active centers, by forming disulphide bridges during the oxidation could be of critical importance. Further studies will be needed to determine whether oxidative stress again ACE can be involved in the genesis of inflammatory vascular pathologies.
体细胞血管紧张素转换酶（ACE）是一种包含两个相似结构域（N和C）的蛋白质，每个结构域都具有一个功能性活性位点。ACE与其天然底物和氧自由基之间的关系正开始被探索。一方面，血管紧张素II可诱导超氧阴离子的产生；另一方面，活化的多核中性粒细胞通过产生自由基改变内皮ACE活性。在本研究中，我们检测了羟基自由基（·OH）对纯化的ACE的影响。·OH使用一种发生器产生：由Lara-Spiral（法国）提供的2,2'-偶氮-双-2-脒基丙烷（GRH）。GRH（3 mM）以时间依赖性方式抑制ACE活性。当ACE与GRH共同孵育4小时时，其活性降低了70%。添加两组·OH清除剂二甲基硫脲（DMTU：0.03至1 mM）或甘露醇+蛋氨酸（20/10 mM），对ACE活性产生剂量依赖性保护作用。为了研究ACE分子中巯基的氧化是否可能参与GRH的作用，研究了巯基还原剂：巯基乙醇和二硫苏糖醇（DTT）的作用。这些化合物产生剂量依赖性且显著的保护作用；巯基乙醇在0.2和0.3 mM时以及DTT在0.1 mM时具有100%的保护作用。还探索了两种天然和结构域特异性底物的水解情况。优先由C结构域裂解的血管紧张素I的水解在与0.3、1和3 mM GRH接触时分别显著（p < 0.01）受到57%、58%和69%的抑制[以每分钟形成的血管紧张素II纳摩尔数/每纳摩尔ACE计，n = 4；35.9 ± 0.6（对照），15.5 ± 2.8（GRH：0.3 mM），15.1 ± 0.5（1 mM），10.9 ± 0.6（3 mM）]。N结构域的优先底物血液调节肽（hp）的水解在0.3 mM GRH时不受影响，在1 mM GRH时受到28%（不显著）的抑制[以每分钟水解的ph纳摩尔数/每纳摩尔ACE计，n = 4；12.6 ± 1.9（对照），14.9（GRH：0.3 mM），8.3 ± 4.0（1 mM）]。这些结果表明·OH影响ACE活性，并提示GRH对C结构域有优先影响。·OH的精确作用位点仍然未知。活性中心附近的半胱氨酸残基在氧化过程中通过形成二硫键可能至关重要。需要进一步研究以确定氧化应激对ACE的影响是否可能参与炎症性血管病变的发生。
