Inactivation of endothelin I by deamidase (lysosomal protective protein).
Deamidase cleaves ester and peptide bonds in various substrates and deamidates protected COOH-terminal amino acids. It preferentially hydrolyzes peptides which contain hydrophobic amino acids in the P1' and/or P1 position. Because the COOH-terminal end of endothelin I contains the hydrophobic sequence -Ile19-Ile20-Trp21-OH, we investigated whether human deamidase, purified from platelets, could inactivate this peptide. We found that deamidase readily cleaved off Trp21 with an acid pH optimum, a Km = 22 microM, a kcat of 1454 min-1, and a kcat/Km of 68 microM-1 min-1. We also found the enzyme to be present in target cells of endothelin, in vascular smooth muscle cells. Extracts of cultured vascular smooth muscle cells cleave both the synthetic fluorescent substrate 5-dimethylaminonaphthalene-1-sulfonyl(Dns)-Phe-Leu-Arg and endothelin I by releasing the COOH-terminal amino acid. The reaction was inhibited by diisopropyl fluorophosphate, benzyloxycarbonyl-Gly-Leu-Phe-CH2Cl, and p-chloromercuribenzenesulfonate, which inhibit the purified deamidase, but not by inhibitors of some other peptidases. The rate of hydrolysis of endothelin I in the soluble, 100,000 x g final supernatant of the homogenized smooth muscle cells was 2.1 mumol/h/mg and 3.1 mumol/h/mg for Dns-Phe-Leu-Arg. Thus, smooth muscles, platelets, and many other tissues which contain the deamidase can inactivate endothelin by cleaving the COOH-terminal tryptophan.
Jackman
H L
HL
Laboratory of Peptide Research, University of Illinois College of Medicine, Chicago 60612.
Morris
P W
PW
Deddish
P A
PA
Skidgel
R A
RA
Erdös
E G
EG
D000581:Amidohydrolases / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D002268:Carboxypeptidases / Q000302:isolation & purification / Q000378:metabolism*; D043402:Cathepsin A; D002478:Cells, Cultured; D016232:Endothelins / Q000037:antagonists & inhibitors / Q000378:metabolism* / Q000494:pharmacology; D006023:Glycoproteins / Q000302:isolation & purification / Q000378:metabolism*; D006168:Guinea Pigs; D006801:Humans; D007082:Ileum / Q000187:drug effects / Q000502:physiology; D066298:In Vitro Techniques; D007700:Kinetics; D008969:Molecular Sequence Data; D009130:Muscle, Smooth / Q000187:drug effects / Q000502:physiology; D009131:Muscle, Smooth, Vascular / Q000201:enzymology; D009842:Oligopeptides / Q000138:chemical synthesis; D011652:Pulmonary Circulation; D051381:Rats; D013379:Substrate Specificity
D016232:Endothelins; D006023:Glycoproteins; D009842:Oligopeptides; D002268:Carboxypeptidases; C468629:CTSA protein, human; D043402:Cathepsin A; D000581:Amidohydrolases