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.
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