Quantitative Measurement of mRNA Levels by RT-PCR

Abstract

The original description of endothelin-1 (ET-1) included the concept that a novel processing enzyme, referred to as endothelin-converting enzyme (ECE), was required for cleavage of the Trp²¹–Val²² bond in the biosynthetic intermediate big ET-1 (1). Initially it was thought that ECE could be a chymotrypsinlike enzyme because of the nature of the peptide bond being cleaved (1). The challenge of identifying ECE was soon taken up by many groups and a variety of different proteolytic activities were proposed as potential ECEs (2). By 1990, studies of endogenous ET-1 synthesis by cultured endothelial cells and investigations of the systemic pressor effect of intravenously administered big ET-1 indicated that the physiological ECE was a phosphoramidon-sensitive enzyme (3–5). Phosphoramidon was also shown to inhibit hydrolysis of exogenous big ET-1 by cultured vascular smooth muscle and endothelial cells (6). The pursuit of an ECE that could selectively hydrolyze big ET-1 and was inhibited by phosphoramidon led quickly to the purification and cloning of a metallopeptidase called endothelin-converting enzyme-1 (ECE-1) (7–9). Shortly after this, a second phosphoramidon-sensitive peptidase, with ∼59% structural homology to ECE-1, was cloned and called ECE-2 (10). ECE-1 and ECE-2 are members of a family of type II integral membrane peptidases that also includes neutral endopeptidase 24.11 and the KELL and PEX proteins (11).