Purification and Characterization of an Endothelin Degradation Enzyme from Rat Kidney

Abstract

A soluble protease which effectively inactivated endothelia-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 in-hibitor, but not by metalloprotease inhibitors or other serine protease inhibitors. The purified enzyme degraded endothelin-1 rapidly; K_M and V_max values were 5.9 μM and 0.40 μmol/mg/min, respectively. It removed the carboxyl terminal tryptophan of endothelia-1 by specifically cleaving the Ile²⁰-Trp²¹ peptide bond, yielding a peptide which was three orders of magnitude less potent than endothelia-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.