Purification and characterization of an endothelin degradation enzyme from rat kidney.
A soluble protease which effectively inactivated endothelin-1 was purified 2,700-fold from rat kidney using DE52 anion exchange, concanavalin A-Sepharose, thiopropyl-Sepharose, and Mono P column chromatography. The overall recovery of enzyme activity was 12%. This enzyme appeared to contain two subunits with molecular weights of 34 and 21 kDa. The molecular weight of the active enzyme complex was estimated to be 82 kDa by gel filtration. It was shown to have a pI between 4.8 and 5.2 and a pH optimum of 5.5. Its activity was inhibited by benzyloxycarbonyl-Phe-AlaCHN2 and p-hydroxymercuribenzoic acid, thiol protease inhibitors, and by phenylmethylsulfonyl fluoride, a serine protease inhibitor, but not by metalloprotease inhibitors or other serine protease inhibitors. The purified enzyme degraded endothelin-1 rapidly; KM and Vmax values were 5.9 microM and 0.40 mumol/mg/min, respectively. It removed the carboxyl terminal tryptophan of endothelin-1 by specifically cleaving the Ile20-Trp21 peptide bond, yielding a peptide which was three orders of magnitude less potent than endothelin-1 in causing contraction of porcine coronary arterial rings. In contrast, proendothelin-1 was not degraded by this enzyme. These results are consistent with published findings that show the clearance rate for proendothelin-1 in the pig to be significantly slower than that for endothelin-1. Our study suggests that this enzyme may play a role in the homeostasis of circulating endothelin-1 and contribute to the regulation of vascular tone.
Deng
A Y
AY
Research Department, CIBA-GEIGY Corp., Summit, NJ 07901.
Martin
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LL
Balwierczak
J L
JL
Jeng
A Y
AY
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002268:Carboxypeptidases / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D016232:Endothelins / Q000378:metabolism*; D066298:In Vitro Techniques; D007668:Kidney / Q000201:enzymology*; D008297:Male; D008970:Molecular Weight; D011480:Protease Inhibitors / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D013379:Substrate Specificity; D013552:Swine; D014661:Vasoconstriction
D000596:Amino Acids; D016232:Endothelins; D011480:Protease Inhibitors; D002268:Carboxypeptidases