CLEAVAGE/SECRETION OF ANGIOTENSIN CONVERTING ENZYME

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Angiotensin-converting enzyme (ACE) is a widely distributed metalloprotease which plays a key role in the control of blood pressure and fluid and electrolyte homeostasis. Although ACE exists primarily as a membrane - bound enzyme, a soluble form is present under normal conditions in blood plasma and other body fluids. To delineate the structural relationship between the cell-bound and the soluble forms of the protein and to determine the modes of their biosynthesis, we have used various expression systems of cloned ACE cDNA. Our studies indicate that ACE is anchored in the cellular plasma membrane through a hydrophobic domain near its C- terminal and its extracellular secretion is achieved by a proteolytic cleavage of this membrane anchoring domain. The responsible protease is a cellular enzyme that cleaves ACE at or near the plasma membrane in a regulated fashion. The exact cleavage site has also been determined. We hypothesize that recognition of specific structural features of ACE by a cell-membrane associated secretase activity is responsible for the extracellular release of this protein. To test this hypothesis, we will determine the role of its intracellular, transmembrane and extracellular domains in the process of cleavage-secretion, by deleting, mutating or exchanging each domain, with the corresponding domain of an uncleavable transmembrane protein. We will define the role of specific amino acid residues at and around the cleavage site by introducing suitable amino acid substitution or deletions in the region. We will determine the role of glycosylation in this process, design and express a cell-bound non- secretable form of ACE and determine its enzymatic properties. We also propose to purify and characterize the ACE-secretase activity which is responsible for this cleavage-secretion process. For this purpose we will develop a cell-free assay system for measuring the ACE-secretase activity, process. For this purpose we will develop a cell-free assay system for measuring the ACE-secretase activity, characterize its enzymatic properties, purify it to homogeneity and develop antibody to it, so t hat we can isolate its cDNA clone in the future. Thus, the proposed project will provide a better understanding of how cell-bound ACE is converted to athe soluble form, how the relative proportion of the two forms is physiologically regulated and whether this conversion has a cell- specificity thereby causing localized ACE action in some tissue. Above and beyond the ACE system, our proposed study, will be the prototype for an important and general mode of biological regulation and will identify the first member of a new class of membrane-bound proteases whose primary role is to selectively cleave and release many biologically important proteins.