ANGIOTENSIN CONVERTING ENZYME GENE EXPRESSION

血管紧张素转换酶（ACE）是一种糖基化的胞外蛋白， 以细胞结合和分泌形式存在。 这种酶负责 用于合成具有多种生理作用的血管紧张素II 包括维持体液和电解质平衡以及血压。 循环中ACE水平升高与糖尿病的发病机制相关 “原发性”高血压、心力衰竭和肾衰竭及抑制剂 这种酶被广泛用于这些疾病的临床管理。 因此，重要的是要了解的生物合成的调控， ACE，其结构和功能。 在本申请中，我们建议 利用分子生物学的强大工具来达到这个目的。 先前 研究表明，ACE有两种同工酶形式，ACEp 和ACET，其以组织特异性方式表达。 ACEp是 在血管内皮细胞和上皮细胞中合成， 肾和肠，而ACET仅在精子细胞中合成。 两 同工酶来源于同一基因， 转录起始位点。 这两种合成的mRNA， 相应的蛋白质，有共同的区域，以及同工酶特异性 地区 我们建议调查的分子机制负责 用于两种ACE mRNA的组织特异性表达。 为此，我们 将研究调节细胞凋亡的顺式作用元件和反式作用因子， 他们的转录。 顺式作用元件将通过 合适的嵌合报道基因在转基因小鼠和 转染的细胞培养。 与这些蛋白结合的反式作用因子 顺式作用元件将通过电泳迁移率变化来鉴定 和足迹分析。 我们亦建议研究 ACE蛋白的生物合成和分泌。 常任和 瞬时转染子将用于此目的。 我们会研究 抑制糖基化和细胞表面蛋白水解活性的作用 对ACE的合成、加工和分泌的影响。 使用位点特异性 诱变，我们将确定所需的特定氨基酸残基 对于酶活性，膜锚定，和切割加工的 蛋白 最后，我们将确定细胞结合的酶的性质， ACE通过表达非分泌形式的ACE。 获得的信息 从最后一个目的，将是有价值的理解本地化行动的 体内的ACE

Angiotensin-converting enzyme (ACE) is a glycosylated ectoprotein which is found in both cell-bound .and secreted forms. This enzyme is responsible for the synthesis of angiotensin II which has diverse physiological effects including maintenance of fluid and electrolyte balance and blood pressure. Elevated circulating levels of ACE are associated with the pathogenesis of 'essential' hypertension, heart failure, and renal failure and inhibitors of this enzyme are widely used for clinical management of these diseases. It is therefore important to understand the regulation of biosynthesis of ACE, its structure and its functions. In this application, we propose to use the powerful tools of molecular biology for this purpose. Previous investigations have shown that there are two isozymic forms of ACE, ACEp and ACET, which are expressed in a tissue specific manner. ACEp is synthesized in vascular endothelial cells and in the epithelial cells of kidney and intestine whereas ACET is synthesized only in sperm cells. Both isozymes originate from the same gene by alternative choice of two transcriptional start sites. The two resultant mRNAs, and the corresponding proteins, have common regions as well as isozyme-specific regions. We propose to investigate the molecular mechanisms responsible for tissue-specific expression of the two ACE mRNAs. For this purpose, we will study the cis-acting elements and trans-acting factors which regulate their transcription. The cis-acting elements will be identified by the expression of suitable chimeric reporter genes ,in transgenic mice and in transfected cells in culture. The trans-acting factors which bind to these cis-acting elements will be identified by electrophoretic mobility shift and footprinting assays. We also propose to ,study the routes of biosynthesis and secretion of the ACE proteins. Both permanent and transient transfectants will be used for this purpose. We will examine the effects of inhibiting glycosylation and cell-surface proteolytic activity on the synthesis, processing, and secretion of ACE. Using site-specific mutagenesis, we will identify the specific amino acid residues necessary for enzyme activity, membrane anchoring, and cleavage-processing of the protein. Finally, we will determine the enzymatic properties of cell-bound ACE by expressing a non-secretable form of ACE. The information obtained from the last aim ,will be valuable for understanding localized action of ACE in the body.