Purification and characterization of endothelin-converting enzyme from rat lung.
Purification and characterization of endothelin-converting enzyme from rat lung.
复制标题
大鼠肺内皮素转换酶的纯化和表征。
DOI：
10.1016/s0021-9258(19)36936-4
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发表时间：
1993
期刊：
影响因子：
--
通讯作者：
K. Tanzawa
中科院分区：
文献类型：
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作者：
Masaaki TakahashiS;Youichi Matsushital;Yasuteru Iijimal;K. Tanzawa
从大鼠肺微粒体中纯化出内皮素转化酶，内皮素转化酶是产生一种有效的血管收缩肽内皮素的关键酶。建立了一种简便、灵敏的125i -内皮素-1受体结合法进行纯化研究。该酶用Triton X-100溶解，并通过小麦胚芽凝集素-琼脂糖、锌螯合Sepharose和Blue Bagarose进行序贯柱层析纯化。纯化酶的电泳分析显示一个M(r) 130,000的蛋白带。高效液相色谱分析表明，纯化后的酶能定量地将大内皮素-1转化为内皮素-1，表明该酶特异性地裂解了Trp21和Val22之间的键。金属螯合剂和磷酰胺对酶有抑制作用，硫罕有和卡托普利无抑制作用。肺内皮素转换酶优先选择大内皮素-1而不是大内皮素-2或-3作为底物，动力学分析表明，大内皮素-1的Michaelis常数为0.20微米，最大速度为3.1 nmol/min。分别是Mg蛋白质。肺内皮素转换酶是一种典型的中性金属蛋白酶，与内皮细胞的酶相似。
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.