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：（改编自申请人摘要）血管紧张素