This report summarizes the recent rapid development of research on neutral endopeptidase 24.11 (enkephalinase; NEP) and on two other metalloenzymes, meprin and endopeptidase 24.15. NEP cleaves a variety of active peptides, including enkephalins, at the amino side of hydrophobic amino acids. The cDNA for human, rat, and rabbit NEP has been cloned and the deduced protein sequences revealed a high degree of homology (93-94%). Site-directed mutagenesis proved that an active site glutamic acid is involved in catalysis and two active site histidines are responsible for binding the zinc cofactor. Although NEP was originally discovered in the kidney, it is widely distributed in the body including specific structures in the central nervous system, lung, male genital tract, and intestine and in neutrophils, fibroblasts, and epithelial cells. In tissues and cells NEP is bound to plasma membrane through a hydrophobic membrane-spanning domain near the NH2 terminus, but it is present in soluble form in urine and blood. In addition to enkephalins, NEP cleaves kinins, chemotactic peptide, atrial natriuretic factor (ANF), and substance P in vivo. NEP in the lung is a major inactivator of substance P, which constricts the airway smooth muscles. Because of the possible involvement of NEP in the metabolism of opioid peptides and the cardiac hormone ANF, orally active inhibitors have been synthesized. Compounds that inhibit both aminopeptidase and NEP were reported to prolong the analgesic effects of enkephalins. Other inhibitors given per os prolonged the renal effects of exogenous ANF. A newly synthesized specific inhibitor of NEP was also active in animal experiments as an analgesic. Studies on the structure and function of NEP should lead to further development of therapeutically applicable inhibitors.

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Abstract-We used the isolated N-and C-domains of the angiotensin I-converting enzyme (N-ACE and C-ACE; ACE; kininase II) to investigate the hydrolysis of the active 1-7 derivative of angiotensin (Ang) II and inhibition by 5-S-5-benzamido-4-oxo-6-phenylhexanoyl-L-proline (keto-ACE). Ang-(1-7) is both a substrate and an inhibitor; it is cleaved by N-ACE at approximately one half the rate of bradykinin but negligibly by C-ACE. It inhibits C-ACE, however, at an order of magnitude lower concentration than N-ACE; the IC 50 of C-ACE with 100 mol/L Ang I substrate was 1.2 mol/L and the K i was 0.13. While searching for a specific inhibitor of a single active site of ACE, we found that keto-ACE inhibited bradykinin and Ang I hydrolysis by C-ACE in approximately a 38-to 47-times lower concentration than by N-ACE; IC 50 values with C-ACE were 0.5 and 0.04 mol/L. Furthermore, we investigated how Ang-(1-7) acts via bradykinin and the involvement of its B 2 receptor. Ang-(1-7) was ineffective directly on the human bradykinin B 2 receptor transfected and expressed in Chinese hamster ovary cells. However, Ang-(1-7) potentiated arachidonic acid release by an ACE-resistant bradykinin analogue (1 mol/L), acting on the B 2 receptor when the cells were cotransfected with cDNAs of both B 2 receptor and ACE and the proteins were expressed on the plasma membrane of Chinese hamster ovary cells. Thus like other ACE inhibitors, Ang-(1-7) can potentiate the actions of a ligand of the B 2 receptor indirectly by binding to the active site of ACE and independent of blocking ligand hydrolysis. This potentiation of kinins at the receptor level can explain some of the well-documented kininlike actions of Ang-(1-7).(Hypertension. 1998;31:912-917.)

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After extracting converting enzyme from a membrane fraction of homogenized human kidney, "enkephalinase" activity was solubilized with Triton X-100. Ion-exchange chromatography resolved two peaks of the "enkephalinase" activity, both of which cleaved Leu5-enkephalin at the Gly3-Phe4 bond. The major "enkephalinase" form was purified 1140-fold to homogeneity with a 14% yield. This homogeneous "enkephalinase" had a specific activity of 46 mumol min-1 mg-1 with Leu5-enkephalin as substrate. The purified enzyme, in addition to hydrolyzing Leu5-enkephalin, cleaved synthetic substrates with protected N- and C-terminal ends. On the basis of the specificity of the enzyme and its inhibition by chelating agents, human "enkephalinase" can be classified as a neutral metalloendopeptidase with a broad substrate specificity. The activity of this neutral endopeptidase with several biologically active peptides was compared to that of homogeneous human kidney converting enzyme. Both enzymes inactivated bradykinin by release of the C-terminal dipeptide but were inhibited differentially by specific inhibitors. Comparison of hydrolysis of bradykinin with that of its protected C-terminal peptide indicated that the neutral endopeptidase is more active toward the larger substrate than is converting enzyme. Although the neutral endopeptidase did not convert angiotensin I to II, it did hydrolyze angiotensin I at Pro7-Phe8 and inactivate angiotensin II by cleavage at the Tyr4-Ile5 bond.

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