Identification of a high-affinity binding protein for a hepta-beta-glucoside phytoalexin elicitor in soybean.
A putative receptor protein for a hepta-beta-glucoside phytoalexin elicitor was identified by photoaffinity labeling of detergent-solubilized proteins from soybean root membranes. Incubation of partially purified beta-glucan-binding proteins with a photolabile 125I-labeled 2-(4-azidophenyl)ethyl-amino conjugate of the heptaglucoside elicitor, followed by irradiation with ultraviolet light (366 nm) resulted in specific labeling of a 70-kDa band in SDS/PAGE. Half-maximal inhibition of the 125I-labeling of the protein band by underivatized hepta-beta-glucoside was achieved by 15 nM heptaglucoside. Analysis of the affinity of radiolabel incorporation into the protein by ligand-saturation experiments, gave an apparent Kd value of 3 nM, in full agreement with the results from radioligand-binding studies. Good correlation was also observed between the amount of radiolabel incorporated into the protein and the binding activity of the fractions obtained at different stages in the purification of heptaglucoside-binding activity. Photoaffinity labeling of proteins purified by glucan-affinity chromatography showed the 70-kDa band as the main component along with weak 125I-labeling of a 100-kDa band. The 70-kDa band was also the major protein visualized by silver staining after SDS/PAGE of this fraction, suggesting that it is the predominant form of the heptaglucoside-binding proteins in detergent-solubilized soybean membranes.
Cosio
E G
EG
Lehrstuhl für Biochemie der Pflanzen, Biologisches Institut II der Universität, Freiburg, Federal Republic of Germany.
Frey
T
T
Ebel
J
J
D000345:Affinity Labels; D002240:Carbohydrate Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D005936:Glucans / Q000138:chemical synthesis / Q000378:metabolism*; D005944:Glucosamine / Q000031:analogs & derivatives / Q000138:chemical synthesis / Q000378:metabolism; D007700:Kinetics; D008024:Ligands; D008565:Membrane Proteins / Q000378:metabolism; D008861:Microsomes / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010936:Plant Extracts / Q000378:metabolism*; D010940:Plant Proteins / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000378:metabolism; D012717:Sesquiterpenes; D013025:Glycine max; D013729:Terpenes; D014466:Ultraviolet Rays; D000094162:Phytoalexins
D000345:Affinity Labels; D005936:Glucans; D008024:Ligands; D008565:Membrane Proteins; D010936:Plant Extracts; D010940:Plant Proteins; D011956:Receptors, Cell Surface; D012717:Sesquiterpenes; D013729:Terpenes; C073685:heptaglucoside; C073684:1-(2-(4-aminophenyl)ethyl)amino-1-(hexaglucosyl)deoxyglucitol; D005944:Glucosamine; D000094162:Phytoalexins