Purification of a novel endothelin-converting enzyme specific for big endothelin-3.
Endothelin-3 (ET-3), a potent vasoactive peptide, is considered to be produced from big ET-3 by endothelin-converting enzyme (ECE) like the other members of the endothelin family (ET-1 and ET-2). We purified a novel ECE from bovine iris microsomes. The purified enzyme, a 140 kDa protein by SDS-PAGE analysis, converted big ET-3 to ET-3 but not big ET-1, with a Km value of 0.14 microM for big ET-3. The conversion to ET-3 was confirmed with sandwich EIA by monoclonal antibodies, the elution profile of HPLC, and intracellular calcium mobilization in CHO-K1 cells expressing recombinant human ET(B) receptors. The conversion activity was inhibited by an inhibitor of neutral endopeptidase 24.11 (NEP) phosphoramidon. These results show that ECE-3 purified from bovine iris is a novel metalloprotease totally different from ECE-1 or ECE-2, in that the enzyme is highly specific for big ET-3.
Hasegawa
H
H
Department of Pharmacology, Kyoto University, Faculty of Medicine, Japan.
Hiki
K
K
Sawamura
T
T
Aoyama
T
T
Okamoto
Y
Y
Miwa
S
S
Shimohama
S
S
Kimura
J
J
Masaki
T
T
D000818:Animals; D016282:Aspartic Acid Endopeptidases / Q000302:isolation & purification* / Q000378:metabolism*; D002417:Cattle; D002829:Choroid / Q000201:enzymology; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D019334:Endothelin-3; D000072396:Endothelin-Converting Enzymes; D016232:Endothelins / Q000378:metabolism*; D005123:Eye / Q000201:enzymology*; D006801:Humans; D007498:Iris / Q000201:enzymology; D007700:Kinetics; D008666:Metalloendopeptidases; D008861:Microsomes / Q000201:enzymology*; D008970:Molecular Weight; D011498:Protein Precursors / Q000378:metabolism*; D012160:Retina / Q000201:enzymology; D013379:Substrate Specificity
D019334:Endothelin-3; D016232:Endothelins; D011498:Protein Precursors; D016282:Aspartic Acid Endopeptidases; D008666:Metalloendopeptidases; C000606923:ECE1 protein, human; C000606924:ECE2 protein, human; D000072396:Endothelin-Converting Enzymes