Effects of the N-terminal sequence of ACE on the properties of its C- domain

Abstract

Angiotensin I-converting enzyme (ACE, kininase II) has 2 active domains (N and C) in a single peptide chain. Because we found its N-domain more stable than its C-domain, we investigated the effect of the amino-terminus of human ACE on the C-domain with a molecular construct expressed in Chinese hamster ovary cells (CHO) cells and transiently in HEK293 cells. This active N-deleted ACE contained only the first 141 amino acids of the human N-domain but not its active center and was linked to the active C-domain containing the transmembrane and cytosolic portions of ACE. The CHO cells were also transfected with human B2 bradykinin receptor. ACE inhibitors (5 nmol/L or 1 μmol/L) augmented bradykinin (100 nmol/L) effects, elevated B2 receptor numbers, and resensitized the receptor desensitized by agonist as measured by arachidonic acid release or [Ca2+](i) mobilization. Arachidonic acid release was mediated by pertussis toxin-sensitive Gα(i), and [Ca2+](i) mobilization was mediated by pertussis-insensitive Gα(q) protein receptor complex. The properties of the construct were compared with wild-type ACE and separate N- and C-domains. The N-deleted ACE differed from wild-type in activation by Cl- and [SO4]2- ions, hydrolysis ratios of substrates (both short synthetic and endogenous peptides) and heat stability. Thus, the N- terminal peptide of ACE affected the characteristics of the C-domain active center. ACE inhibitors acting on N-deleted ACE, which had only a single C- domain active center anchored to plasma membrane, induced cross-talk between the enzyme and the B2 receptor (eg, the inhibitors resensitized the receptor) independent of blocking bradykinin inactivation.