Okumura Yoshiyuki, Matsui Takeshi, Ogawa Kenji, Uchiya Kei-Ichi, Nikai Toshiaki
Department of Microbiology, Faculty of Pharmacy, Meijo University, 150 Yagotoyama, Tempaku-ku, Nagoya, Aichi 468-8503, Japan.
Department of Validation, Matsuurayakugyo Co. Ltd, 24-21 Enjo-chou, Syowa-ku, Nagoya, Aichi 466-0054, Japan.
J Med Microbiol. 2008 Jul;57(Pt 7):803-808. doi: 10.1099/jmm.0.47789-0.
An elastase inhibitor from Aspergillus fumigatus (AFUEI) was isolated, and its biochemical properties and primary structure examined. The inhibitor was purified by column chromatography using DE52 cellulose and Sephadex G-75, and was found to be homogeneous as indicated by a single band following discontinuous PAGE and SDS-PAGE. A molecular mass of 7525.1 Da was observed by matrix-assisted desorption/ionization time-of-flight mass spectroscopy. The elastolytic activity of elastases from A. fumigatus, Aspergillus flavus and human leukocytes was inhibited by AFUEI. However, the elastolytic activity of porcine pancreas elastase, Pseudomonas aeruginosa elastase and elastase from snake venom was not affected by AFUEI. No inhibitory effect of DTT or 2-mercaptoethanol on the elastase inhibitory activity of AFUEI was observed. The amino acid sequence of AFUEI peptides derived from digests utilizing clostripain was determined by Edman sequencing. AFUEI was composed of 68 aa and had a calculated molecular mass of 7526.2 Da. The search for amino acid homology with other proteins demonstrated that aa 1-68 of AFUEI are 100 % identical to aa 20-87 of the hypothetical protein AFUA 3G14940 of A. fumigatus.
从烟曲霉中分离出一种弹性蛋白酶抑制剂（AFUEI），并对其生化特性和一级结构进行了研究。该抑制剂通过使用DE52纤维素和Sephadex G - 75的柱色谱法进行纯化，经不连续聚丙烯酰胺凝胶电泳（PAGE）和十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳（SDS - PAGE）后呈现单一条带，表明其具有均一性。通过基质辅助激光解吸/电离飞行时间质谱法观察到其分子量为7525.1 Da。AFUEI可抑制烟曲霉、黄曲霉和人白细胞弹性蛋白酶的弹性水解活性。然而，猪胰腺弹性蛋白酶、铜绿假单胞菌弹性蛋白酶和蛇毒弹性蛋白酶的弹性水解活性不受AFUEI影响。未观察到二硫苏糖醇（DTT）或2 - 巯基乙醇对AFUEI弹性蛋白酶抑制活性有抑制作用。利用梭菌蛋白酶消化产生的AFUEI肽段的氨基酸序列通过埃德曼测序法确定。AFUEI由68个氨基酸组成，计算分子量为7526.2 Da。与其他蛋白质的氨基酸同源性搜索表明，AFUEI的第1 - 68位氨基酸与烟曲霉假定蛋白AFUA 3G14940的第20 - 87位氨基酸100%相同。
