一种对大内皮素-3具有特异性的新型内皮素转换酶的纯化

Purification of a novel endothelin-converting enzyme specific for big endothelin-3.

作者信息

Hasegawa H, Hiki K, Sawamura T, Aoyama T, Okamoto Y, Miwa S, Shimohama S, Kimura J, Masaki T

机构信息

Department of Pharmacology, Kyoto University, Faculty of Medicine, Japan.

出版信息

FEBS Lett. 1998 May 29;428(3):304-8. doi: 10.1016/s0014-5793(98)00554-7.

DOI:10.1016/s0014-5793(98)00554-7

PMID:9654154

Abstract

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.

摘要

内皮素-3（ET-3）是一种强效血管活性肽，与内皮素家族的其他成员（ET-1和ET-2）一样，被认为是由大内皮素-3经内皮素转换酶（ECE）生成的。我们从牛虹膜微粒体中纯化出一种新型ECE。经SDS-PAGE分析，纯化后的酶是一种140 kDa的蛋白质，它能将大内皮素-3转化为内皮素-3，但不能转化大内皮素-1，对大内皮素-3的Km值为0.14微摩尔。通过单克隆抗体夹心酶免疫分析、高效液相色谱洗脱图谱以及在表达重组人ET（B）受体的CHO-K1细胞中的细胞内钙动员，证实了向内皮素-3的转化。中性内肽酶24.11（NEP）抑制剂磷酰胺可抑制这种转化活性。这些结果表明，从牛虹膜中纯化出的ECE-3是一种与ECE-1或ECE-2完全不同的新型金属蛋白酶，因为该酶对大内皮素-3具有高度特异性。