Purification of a novel endothelin‐converting enzyme specific for big endothelin‐3

FEBS Letters

Shimohama, Shun³  Aoyama, Takuma¹  Hasegawa, Hiroshi¹  Sawamura, Tatsuya²  Hiki, Kazuaki¹  Masaki, Tomoh²  Miwa, Soichi¹  Okamoto, Yasuo¹  Kimura, Jun³ 

[1] Department of Pharmacology, Kyoto University, Faculty of Medicine, Sakyo-ku, Kyoto 606-8315, Japan;National Cardiovascular Centre Research Institute, Osaka 565-0873, Japan;Department of Neurology, Kyoto University, Faculty of Medicine, Sakyo-ku, Kyoto 606-8315, Japan

关键词: Endothelin-3;    Endothelin-converting enzyme;    Enzyme purification;    Bovine iris;    ET;    endothelin;    ECE;    endothelin-converting enzyme;    WGA;    wheat germ agglutinin;    PNA;    peanut agglutinin;    HPLC;    high performance liquid chromatography;    FPLC;    fast protein liquid chromatography;    EIA;    enzyme immunoassay;    PAGE;    polyacrylamide gel electrophoresis;    CHO;    Chinese hamster ovary;   

DOI  :  10.1016/S0014-5793(98)00554-7

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

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 K _m value of 0.14 μM 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.

【 授权许可】

Unknown