Identification of a Kunitz inhibitor from Albizzia kalkora and its inhibitory effect against pest midgut proteases.
A purification protocol, involving water extraction, ammonium sulfate precipitation, Sepharose 4B-trypsin affinity and FPLC Superdex G-75 chromatography, was employed to isolate a trypsin inhibitor from Albizzia kalkora seeds. The inhibitor, which had a molecular mass of 19,768.23 Da, consisted of two disulfide-linked polypeptide chains with approximate molecular mass of 15.5 and 4.5 kDa, respectively. It was stable from pH 2-12 for 24 h, whereas it was unstable either above 80 degrees C for 10 min or under reduced condition over 60 min. The inhibitor, which inhibited trypsin activity with an apparent K (i) of 2.5 x 10(-7) M, had one reactive site involved with a lysine residue. Disulfide linkage and lysine residue were important in maintaining its active conformation. Partial amino acid sequence of the purified protein showed a high degree of homology with various members of the Kunitz inhibitor family. Moreover, trypsin-like proteases from larval Helicoverpa armigera, Spodoptera exigua, and Pieris rapae were inhibited for 85, 57, and 68% respectively, by the inhibitor at 45 microg ml(-1).
Zhou
Jia-Yu
JY
College of Life Science, Sichuan University, Chengdu 610-064, China.
Liao
Hai
H
Zhang
Nian-Hui
NH
Tang
Lin
L
Xu
Ying
Y
Chen
Fang
F
D031289:Albizzia / Q000187:drug effects / Q000378:metabolism*; D000818:Animals; D004064:Digestive System / Q000187:drug effects / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D007313:Insecta / Q000187:drug effects / Q000201:enzymology*; D010447:Peptide Hydrolases / Q000378:metabolism*; D010455:Peptides / Q000302:isolation & purification*; D010940:Plant Proteins / Q000302:isolation & purification*; D011480:Protease Inhibitors / Q000494:pharmacology*; D012639:Seeds / Q000187:drug effects / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics; D017186:Titrimetry
C053865:Kunitz-type protease inhibitor, plant; D010455:Peptides; D010940:Plant Proteins; D011480:Protease Inhibitors; D010447:Peptide Hydrolases