Inactivation of endothelin-1 by an enzyme of the vascular endothelial cells.
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-terminus with hydrophobic amino acids in the P1 or P1' position or both and thereby inactivates endothelin-1, which has a C-terminal sequence of Ile19-Ile20-Trp21-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(6) cells at pH 5.5 at 37 degrees C. Endothelin-1 was completely inactivated by the S3 fraction as determined on rat thoracic aorta strips. The major site of inactivation was the Ile20-Trp21 bond, established by high performance liquid chromatography and by amino acid analysis where the main product was des-Trp21-endothelin-1. The hydrolysis of endothelin-1 (5.9 nmol/min per milligram of protein at pH 5.5 at 23 degrees 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 it.
Laboratory of Peptide Research, University of Illinois College of Medicine, Chicago 60612.
H L
HL
Jackman
P W
PW
Morris
S F
SF
Rabito
G B
GB
Johansson
R A
RA
Skidgel
E G
EG
Erdös
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002268:Carboxypeptidases / Q000378:metabolism*; D043402:Cathepsin A; D003619:Dansyl Compounds / Q000378:metabolism; D016232:Endothelins / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D004730:Endothelium, Vascular / Q000166:cytology / Q000201:enzymology*; D006023:Glycoproteins / Q000378:metabolism*; D009842:Oligopeptides / Q000378:metabolism
D000596:Amino Acids; D003619:Dansyl Compounds; D016232:Endothelins; D006023:Glycoproteins; D009842:Oligopeptides; C043048:dansyl-phenylalanyl-leucyl-arginine; D002268:Carboxypeptidases; D043402:Cathepsin A