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 exogenous 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-terminal fragment (CTF), the effects on porcine and human big ET-1 of each of the purified enzymes were compared in vitro. 2. For E-24.11, the relative rates of hydrolysis were ET-1 > CTF >> big ET-1. The relative half-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 min. 3. For thermolysin the relative rates of hydrolysis were found to be big ET-1 > CTF > ET-1. The relative half-lives for hydrolysis of 3 nmol 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 by RIA. Incubation of big ET-1 (2 nmol) with E-24.11 (40 ng) for 8 h showed that steady state levels of ET-1 were achieved after 4 h indicating that the rate of ET-1 degradation was then equal to the formation of new ET-1. Characterization of the immunoreactivity by h.p.l.c. and RIA confirmed that authentic ET-1 had been produced, but the yield was insufficient for verification by mass spectrometry.5. Both ET-l-like and 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 spectrometry confirmed the production of ET-1 with amounts in the range 120-160 pmol.6. The hydrolysis profile of ET-1 by E-24.11 and thermolysin shows that both enzymes have some common cleavage sites consistent with their similar specificities hydrolysing on the amino side of a hydrophobic residue.7. Thermolysin, for which 3D structural information is available, may represent a better model for endothelin converting enzyme (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.
Murphy
L J
LJ
Department of Biochemistry & Molecular Biology, University of Leeds.
Corder
R
R
Mallet
A I
AI
Turner
A J
AJ
D000818:Animals; D002851:Chromatography, High Pressure Liquid; D019332:Endothelin-1; D016232:Endothelins / Q000378:metabolism*; D006020:Glycopeptides / Q000494:pharmacology*; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D013058:Mass Spectrometry; D015260:Neprilysin / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism; D011498:Protein Precursors / Q000378:metabolism*; D011863:Radioimmunoassay; D013373:Substance P / Q000378:metabolism; D013552:Swine; D013820:Thermolysin / Q000037:antagonists & inhibitors / Q000378:metabolism*
D019332:Endothelin-1; D016232:Endothelins; D006020:Glycopeptides; D010446:Peptide Fragments; D011498:Protein Precursors; D013373:Substance P; D015260:Neprilysin; D013820:Thermolysin; C008890:phosphoramidon