GENERATION BY THE PHOSPHORAMIDON-SENSITIVE PEPTIDASES, ENDOPEPTIDASE-24.11 AND THERMOLYSIN, OF ENDOTHELIN-1 AND C-TERMINAL FRAGMENT FROM BIG ENDOTHELIN-1

1 Phosphoramidon, a potent inhibitor of endopeptidase-24.11 (E-24.11) and thermolysin, has been shown to reduce the hypertensive effect of e xogenous big endothelin-1 (big ET-1) in rats. To examine whether E-24. 11 or thermolysin convert big ET-1 to endothelin-1 (ET-1) and C-termin al fragment (CTF), the effects on porcine and human big ET-1 of each o f the purified enzymes were compared in vitro. 2 For E-24.11, the rela tive rates of hydrolysis were ET-1 > CTF >> big ET-1. The relative hal f-lives for hydrolysis of 3 nmol of each peptide by 200 ng enzyme were : big ET-1 > 24 h; ET-1, 37 min; CTF, 57 min. For comparison, the half -life for hydrolysis of substance P under similar conditions was 2.1 m in. 3 For thermolysin the relative rates of hydrolysis were found to b e big ET-1 > CTF > ET-1. The relative half-lives for hydrolysis of 3 n mol peptide by 50 ng enzyme were: big ET-1, 25 min; ET-1, 56 min; CTF, 47 min. 4 Because the low rate of conversion of big ET-1 to ET-1 by E -24.11 did not yield sufficient ET-1 for h.p.l.c. quantification a RIA specific for ET-1(16-21) was used to study further the hydrolysis of big ET-1 by E-24.11. Incubation of big ET-1 (0.2-2 nmol) with E-24.11 (4-400 ng) generated ET-1 levels of between 1.7 and 33 pmol measured b y RIA. Incubation of big ET-1 (2 nmol) with E-24.11 (40 ng) for 8 h sh owed that steady state levels of ET-1 were achieved after 4 h indicati ng that the rate of ET-1 degradation was then equal to the formation o f new ET-1. Characterization of the immunoreactivity by h.p.l.c. and R IA confirmed that authentic ET-1 had been produced, but the yield was insufficient for verification by mass spectrometry. 5 Both ET-1-like a nd CTF-like peaks were detected at 214 nm when the products of big ET- 1 hydrolysis by thermolysin were resolved by h.p.l.c. RIA and mass spe ctrometry confirmed the production of ET-1 with amounts in the range 1 20-160 pmol. 6 The hydrolysis profile of ET-1 by E-24.11 and thermolys in shows that both enzymes have some common cleavage sites consistent with their similar specificities hydrolysing on the amino side of a hy drophobic residue. 7 Thermolysin, for which 3D structural information is available, may represent a better model for endothelin converting e nzyme (ECE) action than E-24.11 and could be useful for the design of ECE inhibitors. Since E-24.11 can both synthesize and hydrolyse ET-1, the presence of E-24.11 in membrane fractions or in partially purified ECE preparations may produce misleading estimates of ECE activity.