Generation by the phosphoramidon‐sensitive peptidases, endopeptidase‐24.11 and thermolysin, of endothelin‐1 and C‐terminal fragment from big endothelin‐1

摘要: 1 Phosphoramidon, a potent inhibitor of endopeptidase-24.11 (E-24.11) and thermolysin, has been shown to reduce the hypertensive effect exogenous big endothelin-1 (big ET-1) in rats. To examine whether E-24.11 or thermolysin convert ET-1 (ET-1) C-terminal fragment (CTF), effects on porcine human each purified enzymes were compared vitro. 2 For E-24.11, relative rates hydrolysis > CTF >> ET-1. The half-lives for 3 nmol peptide by 200 ng enzyme were: 24 h; ET-1, 37 min; CTF, 57 min. For comparison, half-life substance P under similar conditions was 2.1 min. 3 For found be 50 25 56 47 min. 4 Because low rate conversion did not yield sufficient h.p.l.c. quantification RIA specific (16–21) used study further E-24.11. Incubation (0.2–2 nmol) with (4–400 ng) generated levels between 1.7 33 pmol measured RIA. (2 (40 8 h showed that steady state achieved after 4 indicating degradation then equal formation new Characterization immunoreactivity confirmed authentic had produced, but insufficient verification mass spectrometry. 5 Both ET-1-like CTF-like peaks detected at 214 nm when products resolved spectrometry production amounts range 120–160 pmol. 6 The profile shows both have some common cleavage sites consistent their specificities hydrolysing amino side hydrophobic residue. 7 Thermolysin, which 3D structural information is available, may represent better model endothelin converting (ECE) action than could useful design ECE inhibitors. Since can synthesize hydrolyse presence membrane fractions partially preparations produce misleading estimates activity.