Takahashi M, Matsushita Y, Iijima Y, Tanzawa K
Fermentation Research Laboratories, Sankyo Co., Ltd., Tokyo, Japan.
J Biol Chem. 1993 Oct 5;268(28):21394-8.
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.
内皮素转化酶是一种在强效血管收缩肽内皮素生成过程中的关键酶，它从大鼠肺微粒体中被纯化至同质状态。为了进行纯化研究，开发了一种使用¹²⁵I-内皮素-1受体结合测定的灵敏且便捷的测定方法。该酶用Triton X-100溶解，并通过依次在麦胚凝集素琼脂糖、锌螯合琼脂糖和蓝色琼脂糖柱上进行层析以高产率纯化。对纯化酶的电泳分析显示有一条分子量为130,000的蛋白带。对酶反应的高效液相色谱分析表明，纯化酶能将大内皮素-1定量转化为内皮素-1，这表明该酶特异性地切割了色氨酸²¹和缬氨酸²²之间的键。该酶受到金属螯合剂和磷酰胺脒的抑制，但不受硫氧还蛋白和卡托普利的抑制。肺内皮素转化酶优先选择大内皮素-1而非大内皮素-2或-3作为底物，动力学分析表明，大内皮素-1的米氏常数和最大反应速度分别为0.20微摩尔和3.1纳摩尔/分钟·毫克蛋白。肺内皮素转化酶是一种典型的中性金属蛋白酶，与内皮细胞中的酶相似。
