The Endothelin-Converting Enzyme from Human Umbilical Vein is a Membrane- Bound Metalloprotease Similar to that from Bovine Aortic Endothelial Cells

A phosphoramidon-sensitive, membrane-bound metalloprotease that cleaves big endothelin 1 (big-ET-1) to ET-1 was obtained from human umbilical vein endothelial cells and also from bovine aortic endothelial cells by isolation of plasma-membrane vesicles free of lysosomes. The enzyme was characterized by RIA with an antibody specific for ET-1 and also by reverse-phase HPLC. For both sources, the pH rate profile of the membrane fraction had a very sharp maximum at pH 7.0; little or no activity was seen at more acidic pH values. In contrast, the cytosolic fraction had a major peak at acidic pH values, as well as a broad peak in the neutral region. The activity at pH 7.0 in the membrane fraction was shown by reverse-phase HPLC to produce ET-1 and C-terminal fragment as products. This activity was abolished by phosphoramidon, EDTA, and 1,10-phenanthroline but was not inhibited by pepstatin A, phenylmethylsulfonyl fluoride, soybean trypsin inhibitor, leupeptin, or E-64-consistent with the characteristics of a metalloprotease. These results suggest that this activity is from the physiologically relevant, phosphoramidoninhibitable, endothelin-converting enzyme. The activity found at neutral pH values in the cytosolic fraction was only partially inhibited by EDTA and 1,10-phenanthroline but was not inhibited by phosphoramidon. The membrane-bound endothelin-converting enzyme from human umbilical vein endothelial cells and bovine aortic endothelial cells showed marked similarities, including IC50values for phosphoramidon of 2.7 and 1.8 μM and Kmvalues for big-ET-1 of 45.4 and 20.9 μM, respectively. The apparent molecular mass by gel filtration was ≈300-350 kDa for the enzyme from either source. This report characterizes human endothelin-converting enzyme, which may be an important therapeutic target for cardiovascular disease.