Characterization of a high-affinity binding site for the pea albumin 1b entomotoxin in the weevil Sitophilus.
The toxicity of the pea albumin 1b (PA1b), a 37 amino-acid peptide extracted from pea seeds, for cereal weevils (Sitophilus oryzae, Sitophilus granarius and Sitophilus zeamais) was recently discovered. The mechanism of action of this new entomotoxin is still unknown and potentially involves a target protein in the insect tissues. This work describes the characterization of a high-affinity binding site for PA1b in a microsomal fraction of Sitophilus spp. extracts. Purified PA1b was labeled to a high specific radioactivity (c. 900 Ci.mmol-1) using 125I, and the iodinated ligand was found to be biologically active. Binding of this ligand to the microsomal fraction of S. oryzae extract was found to be saturable and reversible, with an affinity (Kd) of 2.6 nm, and a high maximal binding capacity (Bmax) of 40 pmol.mg-1 of protein. A binding site displaying similar characteristics was detectable in the five susceptible weevils strains tested, as well as in the pea aphid or in the fruit fly. However, no binding activity was detectable in extracts from four S. oryzae strains previously shown to be resistant to the toxin through a recessive monogenic mechanism. Therefore, we suggest that this binding site might be involved in the mechanism of action of PA1b.
Gressent
Frédéric
F
UMR 0203 INRA/INSA de Lyon, BF2I, Biologie Fonctionnelle, Insectes et Interactions, INSA Bâtiment Louis Pasteur, Villeurbanne, France. gressent@jouy.inra.fr
Rahioui
Isabelle
I
Rahbé
Yvan
Y
D000418:Albumins / Q000737:chemistry*; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry; D002462:Cell Membrane / Q000737:chemistry; D001517:Coleoptera; D003545:Cysteine / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D004731:Endotoxins / Q000737:chemistry*; D007700:Kinetics; D008024:Ligands; D008861:Microsomes / Q000378:metabolism; D008969:Molecular Sequence Data; D018532:Pisum sativum / Q000378:metabolism*; D010455:Peptides / Q000737:chemistry; D010940:Plant Proteins / Q000737:chemistry; D011485:Protein Binding; D020033:Protein Isoforms; D013997:Time Factors
D000418:Albumins; D002352:Carrier Proteins; D004731:Endotoxins; D008024:Ligands; C089264:PA1 protein, Glycine max; D010455:Peptides; D010940:Plant Proteins; D020033:Protein Isoforms; D003545:Cysteine