Purification and characterization of a phosphoramidon-sensitive endothelin-converting enzyme in porcine aortic endothelium. OFF.
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 G3000SWXL 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 Km for big endothelin-1 and the corresponding Vmax for endothelin-1 formation were 3.3 +/- 0.3 microM and 0.41 +/- 0.02 mumol/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.
Ohnaka
K
K
Third Department of Internal Medicine, Faculty of Medicine, Kyushu University, Japan.
Takayanagi
R
R
Nishikawa
M
M
Haji
M
M
Nawata
H
H
D000595:Amino Acid Sequence; D000818:Animals; D001011:Aorta; D016282:Aspartic Acid Endopeptidases / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D002413:Cations, Divalent / Q000494:pharmacology; D002462:Cell Membrane / Q000201:enzymology; D002853:Chromatography, Liquid; D015180:Electrophoresis, Gel, Two-Dimensional; D000072396:Endothelin-Converting Enzymes; D004730:Endothelium, Vascular / Q000201:enzymology*; D006020:Glycopeptides / Q000494:pharmacology*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008666:Metalloendopeptidases; D008969:Molecular Sequence Data; D011480:Protease Inhibitors / Q000494:pharmacology; D013379:Substrate Specificity; D013552:Swine
D002413:Cations, Divalent; D006020:Glycopeptides; D011480:Protease Inhibitors; D016282:Aspartic Acid Endopeptidases; D008666:Metalloendopeptidases; D000072396:Endothelin-Converting Enzymes; C008890:phosphoramidon