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

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 hum an ACE on the C-domain with a molecular construct expressed in Chinese hams ter 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 tr ansfected with human B-2 bradykinin receptor. ACE inhibitors (5 nmol/L or 1 mu mol/L) augmented bradykinin (100 nmol/L) effects, elevated B-2 receptor numbers, and resensitized the receptor desensitized by agonist as measured by arachidonic acid release or [Ca2+](i) mobilization. Arachidonic acid re lease was mediated by pertussis toxin-sensitive G(alpha i) and [Ca2+](i) mo bilization was mediated by pertussis-insensitive G(alpha q) protein recepto r 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 i n activation by Cl- and [SO4](2-) ions, hydrolysis ratios of substrates (bo th short synthetic and endogenous peptides) and heat stability. Thus, the N -terminal peptide of ACE affected the characteristics of the C-domain activ e center. ACE inhibitors acting on N-deleted ACE, which had only a single C -domain active center anchored to plasma membrane, induced cross-talk betwe en the enzyme and the B-2 receptor (eg, the inhibitors resensitized the rec eptor) independent of blocking bradykinin inactivation.