SEPARATE ACTIVE DOMAINS OF ANGIOTENSIN CONVERTING ENZYME

血管紧张素转换酶的独立活性结构域

• 批准号: 2031330
• 负责人: ERVIN G ERDOS
• 金额: $ 26.05万
• 依托单位: UNIVERSITY OF ILLINOIS AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-08-01 至 2001-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2031330
• 关键词: ACE inhibitors; CHO cells; X ray crystallography; active sites; enzyme activity; enzyme mechanism; enzyme structure; enzyme substrate complex; human tissue; isozymes; laboratory rabbit; peptidyl dipeptidase A; protein purification; protein structure function; recombinant proteins

DESCRIPTION: (Adapted from the Applicant's Abstract) Angiotensin I-converting enzyme (kininase II; ACE) has two active centers in two homologous domains, named N- or C- domain according to their position in the sequence. This proposal was prompted by the applicants discovery of an ACE consisting of a single intact N-domain (N-ACE). Initial findings indicate that the properties of the two domains grossly differ. The long term objective is to investigate the difference in the functions of the domains and their consequences. The immediate goals are to study the following: I. Enzymatic properties of soluble and membrane-bound N- and C-domains of ACE. Hypothesis: The two domains cleave peptides at different rates. The differences stem from their structures and from their proximity to the cell membrane; the N-domain is furthest from the membrane anchor in the C-domain. Specific Aims: Somatic ACE, germinal ACE (C-domain) and N-ACE will be purified and kinetics of substrate hydrolysis and binding of inhibitors and monoclonal antibodies will be assessed for the soluble enzymes. Some experiments will be done with recombinant somatic ACE, C-domain or N-ACE expressed in Chinese hamster ovary cells and anchored to membrane either by a transmembrane peptide or by a glycosylphosphatidylinositol tail. These studies will show how membrane binding affects the access of the active sites to substrates and inhibitors. II. Structural properties and stability of N- and C- domains of ACE. Hypothesis: There is in somatic ACE a scissible "bridge peptide" between the two domains which, when cleaved by a protease, releases active, stable N-ACE but the C-domain is unstable and is inactivated. The molecular weight and COOH-terminal end of naturally occurring N-ACE or that released by a protease in vitro will be determined by electrospray mass spectrometry. The mode of biogenesis of N-ACE and of inactivation of the C-domain will be studied. Antibodies to bridge peptides will be utilized to establish the site of enzymatic cleavage in the bridge section in naturally occurring forms of N-ACE in biological fluids. Large scale purification of N-ACE will be carried out in order to determine its structure by X-ray crystallography. These studies will probe how the two domains react with peptide hormones and inhibitors and the reason for the greater stability of N-ACE. Ultimately, the experiments should lead to a better understanding of the structure and function of ACE and the therapeutic applications of the inhibitors.

描述：（改编自申请人的摘要）血管紧张素 I-转换酶（激肽酶 II；ACE）在两个 同源结构域，根据其在结构域中的位置命名为 N- 或 C- 结构域 顺序。 该提案是由申请人发现 ACE 引发的 由单个完整的 N 结构域 (N-ACE) 组成。 初步调查结果表明 这两个域的属性截然不同。 长期来看 目的是研究域功能的差异 及其后果。 近期目标是研究以下内容： I. ACE 可溶性和膜结合的 N 和 C 结构域的酶学特性。 假设：这两个结构域以不同的速率切割肽。 这 差异源于它们的结构以及它们与细胞的接近程度 膜; N 结构域距离 C 结构域中的膜锚最远。 具体目标：体细胞 ACE、生发 ACE（C 结构域）和 N-ACE 将被 底物水解和抑制剂结合的纯化和动力学 将评估单克隆抗体的可溶性酶。 一些 实验将使用重组体细胞 ACE、C 结构域或 N-ACE 进行 在中国仓鼠卵巢细胞中表达并通过以下方式锚定在膜上 跨膜肽或糖基磷脂酰肌醇尾。 这些 研究将表明膜结合如何影响活性物质的进入 底物和抑制剂的位点。 二. 结构特性和 ACE N 和 C 结构域的稳定性。 假设：体细胞中存在ACE 两个结构域之间的可剪“桥肽”，当被 一种蛋白酶，释放活性、稳定的 N-ACE，但 C 结构域不稳定， 已停用。 天然产物的分子量和COOH末端 将测定发生的 N-ACE 或体外蛋白酶释放的 N-ACE 通过电喷雾质谱法。 N-ACE和N-ACE的生物发生模式 将研究 C 结构域的失活。 桥肽抗体 将用于建立桥中酶促裂解的位点 生物体液中天然存在的 N-ACE 部分。 大的 将进行N-ACE的规模纯化以确定其 通过X射线晶体学确定结构。 这些研究将探讨两者如何 结构域与肽激素和抑制剂发生反应及其原因 N-ACE 的稳定性更高。 最终，实验应该导致 更好地理解ACE的结构和功能 抑制剂的治疗应用。