SEPARATE ACTIVE DOMAINS OF ANGIOTENSIN CONVERTING ENZYME

描述：（改编自申请人摘要）血管紧张素 I-转化酶（kininase II; ACE）在两个细胞中有两个活性中心。 同源结构域，根据它们在细胞中的位置命名为N-或C-结构域。 顺序 这一建议是由申请人发现的ACE引起的 由单个完整的N结构域（N-ACE）组成。 初步调查表明 这两个领域的性质完全不同 长期 目的是研究不同结构域的功能差异 及其后果 近期目标是研究以下几个方面：一. ACE的可溶性和膜结合N-和C-结构域的酶性质。 假设：两个结构域以不同的速率切割肽。 的 差异源于它们的结构和它们与细胞的接近程度 在C结构域中，N结构域离膜锚最远。 具体目标：体细胞ACE，German ACE（C-结构域）和N-ACE将被 纯化和底物水解动力学和抑制剂的结合， 将评估单克隆抗体的可溶性酶。 一些 将用重组体细胞ACE、C-结构域或N-ACE进行实验 在中国仓鼠卵巢细胞中表达，并通过 跨膜肽或糖基磷脂酰肌醇尾。 这些 研究将显示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-结构域稳定性。 假设：体细胞ACE 在两个结构域之间的可切割的“桥肽”，当被 蛋白酶，释放活性，稳定的N-ACE，但C-结构域是不稳定的， 是不活跃的 天然产物的分子量和羧基末端 将测定存在的N-ACE或由蛋白酶在体外释放的N-ACE 通过电喷雾质谱。 N-ACE的生物发生模式和 将研究C结构域的失活。 桥肽抗体 将用于在桥中建立酶促切割位点 在生物液体中天然存在的N-ACE形式。 大 将进行N-ACE的规模纯化，以测定其 由X射线晶体学构造。 这些研究将探讨这两个 结构域与肽激素和抑制剂反应， N-ACE的稳定性更高。 最终，这些实验应该会导致 更好地了解ACE的结构和功能， 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.