Specific, high affinity binding sites for an antifungal plant defensin on Neurospora crassa hyphae and microsomal membranes

被引:89
作者
Thevissen, K
Osborn, RW
Acland, DP
Broekaert, WF
机构
[1] Katholieke Univ Leuven, FA Janssens Lab Genet, B-3001 Heverlee, Belgium
[2] Zeneca Agrochem, Jealotts Hill Res Stn, Bracknell RG42 6ET, Berks, England
关键词
D O I
10.1074/jbc.272.51.32176
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
Hs-AFP1 an antifungal plant defensin from seed of the plant Heuchera sanguinea, was radioactively labeled using t-butoxycarbonyl-[S-35]L-methionine N-hydroxysuccinimidyl ester, resulting in a S-35-labeled peptide with unaltered antifungal activity. [S-35]Hs-AFP1 was used to assess binding on living hyphae of the fungus Neurospora crassa. Binding of [S-35]Hs-AFP1 was found to be competitive, reversible, and saturable with an apparent K-d of 29 nM and a B-max of 1.4 pmol/mg protein. [S-35]Hs-AFP1 also bound specifically and reversibly to microsomal membranes derived hom N. crassa hyphae with a K-d of 27 nM and a B-max of 102 pmol/mg protein. The similarity in K-d value between binding sites on hyphae and microsomes indicates that Hs-AFP1 binding sites reside on the plasma membrane. Binding of [S-35]Hs-AFP1 to both hyphae and microsomal membranes could be competed to some extent by four different structurally related plant defensins but not by various structurally unrelated antimicrobial peptides. In addition, an inactive single amino acid substitution variant of the antifungal plant defensin Rs-AFP2 from Raphanus sativus seed was also unable to displace [S-35]Hs-AFP1 from its binding sites, whereas Rs-AFP2 itself was able to compete with [S-35]Hs-AFP1.
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收藏
页码:32176 / 32181
页数:6
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