Biochemical properties and primary structure of elastase inhibitor AFUEI from Aspergillus fumigatus.
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.
Okumura
Yoshiyuki
Y
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.
Matsui
Takeshi
T
Department of Microbiology, Faculty of Pharmacy, Meijo University, 150 Yagotoyama, Tempaku-ku, Nagoya, Aichi 468-8503, Japan.
Ogawa
Kenji
K
Department of Pulmonary Medicine, Higashi Nagoya National Hospital, 5-101 Umemorizaka, Meito-ku, Nagoya, Aichi 465-8620, Japan.
Uchiya
Kei-Ichi
KI
Department of Microbiology, Faculty of Pharmacy, Meijo University, 150 Yagotoyama, Tempaku-ku, Nagoya, Aichi 468-8503, Japan.
Nikai
Toshiaki
T
Department of Microbiology, Faculty of Pharmacy, Meijo University, 150 Yagotoyama, Tempaku-ku, Nagoya, Aichi 468-8503, Japan.
D000595:Amino Acid Sequence; D001232:Aspergillus fumigatus / Q000737:chemistry / Q000201:enzymology / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D005656:Fungal Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D019272:Leukocyte Elastase / Q000037:antagonists & inhibitors; D008969:Molecular Sequence Data; D010196:Pancreatic Elastase / Q000037:antagonists & inhibitors*; D011480:Protease Inhibitors / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D017422:Sequence Analysis, DNA; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods
D005656:Fungal Proteins; D011480:Protease Inhibitors; D010196:Pancreatic Elastase; D019272:Leukocyte Elastase