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Volume 272, Number 51, Issue of December 19, 1997 pp. 32176-32181

Specific, High Affinity Binding Sites for an Antifungal Plant Defensin on Neurospora crassa Hyphae and Microsomal Membranes

(Received for publication, July 8, 1997, and in revised form, October 9, 1997)

Karin Thevissen , Rupert W. Osborn ^¶ , David P. Acland ^¶ and Willem F. Broekaert

From the  F. A. Janssens Laboratory of Genetics, Katholieke Universiteit Leuven, K. Mercierlaan 92, B-3001 Heverlee, Belgium and ^¶ Zeneca Agrochemicals, Jealott's Hill Research Station, Bracknell, Berkshire RG42 6ET, United Kingdom

Hs-AFP1, an antifungal plant defensin from seed of the plant Heuchera sanguinea, was radioactively labeled using t-butoxycarbonyl-[³⁵S]L-methionine N-hydroxysuccinimidyl ester, resulting in a ³⁵S-labeled peptide with unaltered antifungal activity. [³⁵S]Hs-AFP1 was used to assess binding on living hyphae of the fungus Neurospora crassa. Binding of [³⁵S]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. [³⁵S]Hs-AFP1 also bound specifically and reversibly to microsomal membranes derived from 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 [³⁵S]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 [³⁵S]Hs-AFP1 from its binding sites, whereas Rs-AFP2 itself was able to compete with [³⁵S]Hs-AFP1.

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