SEPARATE ACTIVE DOMAINS OF ANGIOTENSIN CONVERTING ENZYME

描述：(改编自申请者摘要)血管紧张素 I-转换酶(Kininase II；ACE)由两个活性中心组成 同源结构域，根据它们在 序列。这项提议是由申请者发现的ACE引起的 由单个完整的N-结构域(N-ACE)组成。初步调查结果表明 这两个域的性质有很大的不同。从长远来看 目的是调查不同领域的功能差异 以及他们的后果。近期目标是研究以下内容：一. 血管紧张素转换酶可溶性和膜结合N-和C-结构域的酶学性质。 假设：这两个区域以不同的速度裂解多肽。这个 差异源于它们的结构和它们与细胞的接近程度 膜；N-结构域与C-结构域中的膜锚距离最远。 具体目标：体细胞ACE、生发ACE(C结构域)和N-ACE将 底物水解物和抑制剂结合的纯化和动力学 将对单抗进行可溶酶的评估。一些人 将使用重组体细胞ACE、C结构域或N-ACE进行实验 在中国仓鼠卵巢细胞中表达并锚定到膜上 一种跨膜肽或由糖基磷脂酰肌醇尾巴组成。这些 研究将显示膜结合如何影响活性物质的访问 底物和抑制剂的位置。二、结构特性和 血管紧张素转换酶N区和C区的稳定性。假设：体细胞中存在血管紧张素转换酶 两个结构域之间的一种可剪切的“桥肽”，当被切割时 一种蛋白水解酶，释放活性稳定的N-ACE，但C-结构域不稳定 已停用。天然产物的相对分子质量和羧基末端 将确定发生的N-ACE或由体外酶释放的N-ACE 用电喷雾质谱仪测定。N-血管紧张素转换酶和环磷酰胺的生物发生模式 将对C结构域的失活进行研究。抗桥蛋白多肽抗体 将被用来确定桥中的酶切割位置 生物液中天然存在的N-血管紧张素转换酶部分。大型 将对N-ACE进行规模纯化，以确定其 用X-射线结晶学分析了其晶体结构。这些研究将探索两者是如何 结构域与多肽激素和抑制物反应，以及 N-ACE的稳定性更高。最终，这些实验应该会导致一种 更好地了解ACE的结构和功能以及 这些抑制剂的治疗应用。

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.