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β-Glucan-binding protein
Reconstitution
Lipid
Soybean
Abstract:
In temperature-induced Triton X-114 phase separation experiments the β-glucan elicitor-binding site from soybean (Glycine max L.) root membranes was identified as (a) hydrophobic membrane protein(s). The Zwittergent 3-12-solubilized β-glucan-binding proteins were incorporated into lipid vesicles by the detergent-dilution procedure. Reconstituted binding proteins were functional in that binding of the hepta-β-glucoside ligand was saturable, reversible and of high affinity (Kd=6–7 nM). Competition studies using β-glucans with different degrees of polymerization (DP 7–15; DP 15–25) showed effective displacement of the radioligand from the binding site whereas β-glucan fragments with DP <7 were ineffective. The total amount of reconstituted binding activity was dependent on the acyl chain length of the phospholipids used for the reconstitution with a preference for decanoic (C10) and dodecanoic (C12) chains. Restored ligand binding was maximally 37% as compared to the former detergent-solubilized binding activity. The presence of a lipid environment stabilized the purified β-glucan-binding proteins.
