Inactivation of Endothelin-1 by an Enzyme of the Vascular

We previously investigated the inactivation of endothelin-1 by deamidase (lysosomal protective protein), present in many cells, including vascular smooth muscle cells. This enzyme, which we originally purified from human platelets, preferentially hydrolyzes peptides at the C-termlnus with hydrophobic amino adds in the Pi or P, ' position or both and thereby inactivates endothelin-1, which has a C-tenninal sequence of Ile"-IleM-Trp2l-OH. We tested for the presence of deamidase in cultured bovine aortic endothelial cells. The final supernatant of the homogenized cells (S3) cleaved the deamidase substrate dansyl-Phe-Leu-Arg at a rate of 1-3 nmol/min per 10 * cells at pH 5.5 at 37°C. Endothelin-1 was completely inactivated by the S3 fraction as determined on rat thoracic aorta strips. The major site of inactivation was the Ile^-Trp21 bond, established by high performance liquid chromatography and by amino acid analysis where the main product was des-Trp21-endothelin-l. The hydrolysis of endothelin-1 (5.9 nmol/min per milligram of protein at pH 5.5 at 23°C) by S3 was blocked mainly by inhibitors of deamidase, including diisopropyl fluorophosphate, but not by inhibitors of some other peptidases. This is the first report of a novel pathway of endothelin-1 metabolism in endothelial cells. Thus, endothelial cells, besides being the source of endothelin-1, contain an enzyme that inactivates i t (Hypertension 1993^1:925-928) KEY WORDS • cathepsin A • deamidase • endothelium • high performance liquid chromatograph