Purification and characterization of endothelin-converting enzyme from rat lung.
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.
Takahashi
M
M
Fermentation Research Laboratories, Sankyo Co., Ltd., Tokyo, Japan.
Matsushita
Y
Y
Iijima
Y
Y
Tanzawa
K
K
D000818:Animals; D016282:Aspartic Acid Endopeptidases / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D001667:Binding, Competitive; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D000072396:Endothelin-Converting Enzymes; D016232:Endothelins / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008168:Lung / Q000201:enzymology*; D008666:Metalloendopeptidases; D008861:Microsomes / Q000201:enzymology; D051381:Rats; D013379:Substrate Specificity
D016232:Endothelins; D016282:Aspartic Acid Endopeptidases; D008666:Metalloendopeptidases; D000072396:Endothelin-Converting Enzymes