Purification and characterization of a phosphoramidon-sensitive endothelin-converting enzyme in porcine aortic endothelium

An enzyme that catalyzes the conversion of big endothelin-1 to endothelin- 1, designated as endothelin-converting enzyme, was solubilized with Lubrol PX from the membrane fraction of porcine aortic endothelium and was purified by sequential chromatography on DEAE-agarose, Ricinus communis agglutinin 120- agarose, peanut agglutinin-agarose, Mono Q, and TSK G3000SW(XL) columns. Approximately 12,000-fold purification of the membrane fraction enzyme was achieved. The purified enzyme had a very narrow neutral pH optimum and was inhibited by EDTA, 1,10-phenanthroline, phosphoramidon, and low concentrations of some divalent cations (Cu²⁺, Zn²⁺, Co²⁺, Fe²⁺) but not by thiorphan. Addition of Zn²⁺ was most effective for the restoration of the EDTA-inactivated enzyme. The purified enzyme showed the highest affinity for big endothelin-1 among big endothelin isopeptides, and the K(m) for big endothelin-1 and the corresponding V(max) for endothelin-1 formation were 3.3 ± 0.3 μM and 0.41 ± 0.02 μmol/min/mg of protein, respectively. The carboxyl-terminal sequence from His²⁷ to Gly³⁴ and Trp²¹ was essential for recognition by this enzyme, while the presence of the amino- terminal loop structure reduced the hydrolysis rate. The purified enzyme showed an isoelectric point of 4.1. The molecular mass was estimated to be 131 kDa by sucrose density gradient centrifugation, and a value of 120 kDa was obtained by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicating that endothelin-converting enzyme is a monomeric glycoprotein.