Functional reconstitution of beta-glucan elicitor-binding activity upon incorporation into lipid vesicles

In temperature-induced Triton X-114 phase separation experiments the beta-g lucan elicitor-binding site from soybean (Glycine max L.) root membranes wa s identified as (a) hydrophobic membrane protein(s). The Zwittergent 3-12-s olubilized beta-glucan-binding proteins were incorporated into lipid vesicl es by the detergent-dilution procedure. Reconstituted binding proteins were functional in that binding of the hepta-beta-glucoside ligand was saturabl e, reversible and of high affinity (K-d=6-7 nM). Competition studies using P-glucans with different degrees of polymerization (DP 7-15; DP 15-25) show ed effective displacement of the radioligand from the binding site whereas beta-glucan fragments,vith DP <7 were ineffective. The total amount of reco nstituted binding activity was dependent on the acyl chain length of the ph ospholipids 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 presenc e of a lipid environment stabilized the purified beta-glucan-binding protei ns. (C) 1999 Federation of European Biochemical Societies.