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 s anguinea, was radioactively labeled using t-butoxycarbonyl-[S-35]L-met hionine N-hydroxysuccinimidyl ester, resulting in a S-35-labeled pepti de with unaltered antifungal activity. [S-35]Hs-AFP1 was used to asses s 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-3 5]Hs-AFP1 also bound specifically and reversibly to microsomal membran es 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 micro somal membranes could be competed to some extent by four different str ucturally related plant defensins but not by various structurally unre lated antimicrobial peptides. In addition, an inactive single amino ac id substitution variant of the antifungal plant defensin Rs-AFP2 from Raphanus sativus seed was also unable to displace [S-35]Hs-AFP1 from i ts binding sites, whereas Rs-AFP2 itself was able to compete with [S-3 5]Hs-AFP1.