Purification and characterization of endothelin-converting enzyme from rat lung.

M Takahashi,Y Matsushita,Y Iijima,K Tanzawa

doi:10.1016/s0021-9258(19)36936-4

Abstract

Endothelin-converting enzyme, a key enzyme in the production of a potent vasoconstricting peptide, endothelin, was purified to homogeneity from rat lung microsomes. A sensitive and convenient assay method using the 125I-endothelin-1 receptor binding assay was developed for purification studies. The enzyme was solubilized with Triton X-100 and was purified at high yield by sequential column chromatography on wheat germ agglutinin-agarose, zinc-chelating Sepharose, and Blue Bagarose. Electrophoretic analysis of the purified enzyme revealed one protein band with M(r) 130,000. High performance liquid chromatographic analysis of the enzyme reaction revealed that purified enzyme quantitatively converted big endothelin-1 to endothelin-1, indicating that the enzyme specifically cleaved the bond between Trp21 and Val22. The enzyme was inhibited by metal chelators and phosphoramidon, but not by thiorphan and captopril. Lung endothelin-converting enzyme preferred big endothelin-1 to big endothelin-2 or -3 as a substrate, and kinetic analysis revealed that the Michaelis constant and a maximal velocity for big endothelin-1 were 0.20 microM and 3.1 nmol/min.mg protein, respectively. Lung endothelin-converting enzyme is a typical neutral metalloproteinase and is similar to the enzyme from endothelial cells.

Highlights

Lized with Triton X-100 anwd as purifiedat high yield by sequential column chromatographyon wheat germ ag
Standard displacement curves of lZ5I-ET-l specific proteases that degraded big ET-1 and ET-1 were rebinding to SPA microsphere by unlabeled ET-1, ET-2and ET-3 moved during this step and that the removal of nonspecific were prepared for the quantification of produced ET in the testproteases caused the high yield.The active fracsample (Fig. 1).The Amersham ET-1 SPA receptor binding tions from the zinc-chelating Sepharose column were loaded assay system employsporcine lung membranes, which pre- onto a Blue B-agarose column
Purification of lung ECEbyWGA-agarose (A), zincchelatingSepharose( B )and Blue B-agarose (0.Aliquots of each fraction were assayedfor endothelin-converting enzyme (ECE) activity (0)and for protein concentration (0)A., the solubilized protein was applied to a Wheat germ agglutinin (WGA)-agarose column

Summary

RESULTS

From the WGA-agarose columnwere subsequently applied to a Development of 1251-E-ET-Cl ompetition Assayfor the Measure- zinc-chelatingSepharose column. ECEactivity passed through ment of ECE Actiuity-The lZ5I-ET-l competition assay de- the zinc-chelating Sepharose column with an additional %fold scribed under “Experimental Procedures” was developedfor purification (Fig. 3B). The pooled fractions from the zinc-chethe determination of ECE activity This assay method is based lating Sepharose column showed linear time-dependent ECE on the competition for binding to the ET receptors in SPA mi- activity, and the apparent recovery at this step was 184%. Standard displacement curves of lZ5I-ET-l specific proteases that degraded big ET-1 and ET-1 were rebinding to SPA microsphere by unlabeled ET-1, ET-2and ET-3 moved during this step and that the removal of nonspecific were prepared for the quantification of produced ET in the testproteases caused the high yield (over 100%).The active fracsample (Fig. 1).The Amersham ET-1 SPA receptor binding tions from the zinc-chelating Sepharose column were loaded assay system employsporcine lung membranes, which pre- onto a Blue B-agarose column. ET-1 was incubated in 100 pl of ECE assay buffer containing 30 pg of microsomalprotein (01, 15 pg of supernatant of solubilized microsomes (01, or 15 pg of supernatant and 10 pg/ml phosphoramidon (A).The amount of formed ET-1 was determined by the 1261-ET-1competition assay

Specific activity"

Thiorphan CaDtoDd

Findings

DISCUSSION