Specific, high affinity binding sites for an antifungal plant defensin on Neurospora crassa hyphae and microsomal membranes.
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 Kd of 29 nM and a Bmax of 1.4 pmol/mg protein. [35S]Hs-AFP1 also bound specifically and reversibly to microsomal membranes derived from N. crassa hyphae with a Kd of 27 nM and a Bmax of 102 pmol/mg protein. The similarity in Kd 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.
Thevissen
K
K
F. A. Janssens Laboratory of Genetics, Katholieke Universiteit Leuven, K. Mercierlaan 92, B-3001 Heverlee, Belgium.
Osborn
R W
RW
Acland
D P
DP
Broekaert
W F
WF
D000935:Antifungal Agents / Q000378:metabolism*; D001665:Binding Sites; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone / Q000494:pharmacology; D023082:Defensins; D006868:Hydrolysis; D007425:Intracellular Membranes / Q000187:drug effects / Q000378:metabolism; D008861:Microsomes / Q000378:metabolism*; D009492:Neurospora crassa / Q000378:metabolism*; D010940:Plant Proteins / Q000378:metabolism*; D010944:Plants / Q000378:metabolism*; D013462:Sulfur Radioisotopes; D014475:Uncoupling Agents / Q000494:pharmacology
D000935:Antifungal Agents; D023082:Defensins; D010940:Plant Proteins; D013462:Sulfur Radioisotopes; D014475:Uncoupling Agents; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone