NATURALLY-OCCURRING ACTIVE N-DOMAIN OF HUMAN ANGIOTENSIN I-CONVERTINGENZYME

Angiotensin I-converting enzyme (ACE, kininase II) is a single-chain p rotein containing two active site domains (named N- and C-domains acco rding to position in the chain). ACE is bound to plasma membranes by i ts C-terminal hydrophobic transmembrane anchor. Ileal fluid, rich in A CE activity, obtained from patients after surgical colectomy was used as the source. Column chromatography, including modified affinity chro matography on lisinopril-Sepharose, yielded homogeneous ACE after only a 45-fold purification. N-terminal sequencing of heal ACE and partial sequencing of CNBr fragments revealed the presence of an intact N ter minus but only a single N-domain active site, ending between residues 443 and 559. Thus, ileal-fluid ACE is a unique enzyme differing from t he widely distributed two-domain somatic enzyme or the single C-domain testicular (germinal) ACE. The molecular mass of heal ACE is 108 kDa and when deglycosylated, the molecular mass is 68 kDa, indicating exte nsive glycosylation (37% by weight). In agreement with the results rep orted with recombinant variants of ACE, the heal enzyme is less Cl- de pendent than somatic ACE; release of the C-terminal dipeptide from a p eptide substrate was optimal in only 10 mM Cl-. In addition to hydroly zing at the C-terminal end of peptides, deal ACE efficiently cleaved t he protected N-terminal tripeptide from the luteinizing hormone-releas ing hormone and its congener 6-31 times faster, depending on the Cl- c oncentration, than the C-domain in recombinant testicular ACE, Thus we have isolated an active human ACE consisting of a single N-domain. We suggest that there is a bridge section of about 100 amino acids betwe en the active N- and C-domains of somatic ACE where it may be proteoly tically cleaved to liberate the active N-domain. These findings have p otential relevance and importance in the therapeutic application of AC E inhibitors.