Defense proteins from seed of Cassia fistula include a lipid transfer protein homologue and a protease inhibitory plant defensin

被引:104
作者
Wijaya, R
Neumann, GM
Condron, R
Hughes, AB
Polya, GM [1 ]
机构
[1] La Trobe Univ, Dept Biochem, Bundoora, Vic 3083, Australia
[2] La Trobe Univ, Dept Chem, Bundoora, Vic 3083, Australia
基金
澳大利亚研究理事会;
关键词
Cassia fistula; seeds; protease inhibitor; defensin;
D O I
10.1016/S0168-9452(00)00348-4
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
A novel trypsin inhibitor was extracted from the seeds of Cassia fistula by a process successively involving soaking seeds in water, extraction of the seeds in methanol, and extraction of the cell wall material at high ionic strength. The protease inhibitor (PI) was subsequently purified by chromatography on carboxymethylcellulose, gel filtration and reversed phase HPLC (RP-HPLC). Electrospray ionization mass spectrometry (ESMS) of the oxidized from of the PI yielded an average molecular mass of 5458.6 +/- 0.8 Da. Edman sequencing of the PI yielded a full-length 50 amino acid sequence inferred to contain eight cysteines and with a calculated average molecular mass (fully oxidized form) of 5459.3 Da, in agreement with the observed mass. The C. fistula seed PI is homologous to the family of plant defensins (gamma -thionins), which have four disulfide linkages at highly conserved locations. The C. fistula PI inhibits trypsin (IC50 2 muM), and is the first known example of a plant defensin with protease inhibitory activity, suggesting a possible additional function for some members of this class of plant defensive proteins. C. fistula seeds also contain a 9378 Da lipid transfer protein (LTP) homologue, other LTPs, a 7117 Da protein copurifying with PI activity and a 5144 Da defensin which does not inhibit trypsin. The complete sequence of the 5144 Da defensin was determined by Edman sequencing, yielding a calculated average molecular mass (oxidized form) of 5144.1 Da, in agreement with the mass observed by ESMS. The likely trypsin inhibitory residue on the 5459 Da defensin is Lysine-25, the corresponding amino acid being Tyrosine-25 in the homologous 5144 Da defensin that is not a trypsin inhibitor. (C) 2000 Elsevier Science Ireland Ltd. All rights reserved.
引用
收藏
页码:243 / 255
页数:13
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