Incubation of endothelins (ETs) with bovine kidney neutral endopeptidase (NEP) resulted in a selective two-step degradation with loss of biochemical activity. The Km of the enzyme indicated high-affinity binding, and hydrolysis was completely inhibited by phosphoramidon. The first step was nicking of the Ser5-Leu' bond, followed by cleavage at the amino side of ile". The nicked peptide exhibited biochemical activities comparable to those of the intact peptide-i.e., binding to the ET receptor, induction of inositol phospholipid hydrolysis, and toxicity. The twice-cleaved product was inactive. The sarafotoxins (SRTXs) were more resistant than the ETs to NEP: for example, the half-time for ET-1 was 41 hr, while it was =4 hr for SRTX-b and even higher for SRTX-c. These in vitro rmdings may indicate a regulatory role of NEP (or similar enzymes) in the physiological inactivation of ETs. They might also help to explain why under certain physiological conditions ETs may be less toxic than SRTXs.The endothelins (ETs) and the sarafotoxins (SRTXs) are two classes of a recently discovered family of peptides containing 21 amino acids (reviewed in refs. 1 and 2). The ETs (isolated from mammalian systems) and the SRTXs (from snake venom) are among the most potent vasoconstrictor agents known (refs. 1 and 2 and references therein) and are highly lethal (3). The two classes of peptides show a considerable sequence homology: they each possess four cysteinyl residues, and "'60%o of the 21 amino acid residues are common to both. Since ET-1 was described, three other ETs have been detected and are designated ET-2, ET-3 (4), and (Vic) (5) (Fig. 1). Also, four SRTXs-SRTX-a, SRTX-b, SRTX-c, and SRTX-d (6)-have been characterized so far (Fig. 1). The four cysteinyl residues in the ETs and in the SRTXs are interconnected by disulfide bridges between positions 1 and 15 and 3 and 11, forming an intramolecular loop structure (see diagram in Fig. 1 (19). The substrates cleaved by NEP appear to be restricted to peptides of molecular mass ==3 kDa (18,19). These considerations prompted us to examine whether the ETs and/or the SRTXs might be good substrates for NEP. The effect of the enzyme can be evaluated by measuring enzyme-associated changes in the biochemical activities of the peptides, such as binding to the receptor, induction of inositol phospholipid hydrolysis, immunogenicity (20, 21), and lethality. As shown in this in vitro study, NEP inactivates ETs as a result of nicking the bond at Ser5-Leu6 followed by rapid cleavage at the amino side of Ile"9. Under identical experimental conditions, NEP also inactivated SRTXs, but at a much slower rate. MATERIALS AND METHODSET-1, ET-2, ET-3, and ET-4 (Vic) were purchased from American Peptide (Santa Clara, CA). SRTX-b and SRTX-c were those used in a previous study (20). 125I-labeled ET-1 (125I-ET-1) was prepared by iodination with Enzymobeads (Bio-Rad) and was purified as described (8). Bovine kidney NEP (0.4 mg/ml) was purified by Triton X-100 extraction followed by DEAE-Sepharose Fast...