Functional reconstitution of beta-glucan elicitor-binding activity upon incorporation into lipid vesicles.
In temperature-induced Triton X-114 phase separation experiments the beta-glucan elicitor-binding site from soybean (Glycine max L.) root membranes was identified as (a) hydrophobic membrane protein(s). The Zwittergent 3-12-solubilized beta-glucan-binding proteins were incorporated into lipid vesicles by the detergent-dilution procedure. Reconstituted binding proteins were functional in that binding of the hepta-beta-glucoside ligand was saturable, reversible and of high affinity (K(d)=6-7 nM). Competition studies using beta-glucans with different degrees of polymerization (DP 7-15; DP 15-25) showed effective displacement of the radioligand from the binding site whereas beta-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 beta-glucan-binding proteins.
Mithöfer
A
A
Botanisches Institut der Universität, Munich, Germany. amithoef@botanik.biologie.uni-muenchen.de
Ebel
J
J
D001667:Binding, Competitive; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism*; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D003902:Detergents; D005936:Glucans / Q000737:chemistry / Q000378:metabolism*; D037102:Lectins; D008861:Microsomes / Q000737:chemistry / Q000378:metabolism; D017830:Octoxynol; D010713:Phosphatidylcholines / Q000737:chemistry / Q000378:metabolism; D010940:Plant Proteins / Q000737:chemistry / Q000378:metabolism*; D011092:Polyethylene Glycols; D011510:Proteolipids / Q000737:chemistry / Q000378:metabolism*; D012995:Solubility; D013025:Glycine max
D002352:Carrier Proteins; D003902:Detergents; D005936:Glucans; D037102:Lectins; D010713:Phosphatidylcholines; D010940:Plant Proteins; D011510:Proteolipids; C037937:glucan-binding proteins; C073685:heptaglucoside; C020485:proteoliposomes; C015153:1,2-dilauroylphosphatidylcholine; D011092:Polyethylene Glycols; D017830:Octoxynol; C010615:Nonidet P-40