Affinity purification and characterization of a binding protein for a hepta-β-glucoside. Phytoalexin elicitor in soybean

Thomas Frey, Eric G. Cosio, Jorgen Ebel

Producción científica: Contribución a una revistaArtículorevisión exhaustiva

43 Citas (Scopus)


A hepta-β-glucoside elicitor of phytoalexin accumulation binds with high affinity to the plasma membrane of soybean (Glycine max) cells. One component of the elicitor-binding sites is a protein with an apparent Mr of 70 000 in SDS-PAGE as recently identified by photoaffinity labelling using a photosensitive 125I-labelled 2-(4-azidophenyl) ethylamine conjugate of the heptaglucoside. Heptaglucoside-binding activity was solubilized using the zwitterionic detergent, Zwittergent 3-12, and purified by ion-exchange chromatography on Q Sepharose and by affinity chromatography. The affinity adsorbent consisted of a Phytophthora megasperma branched (1 → 3, 1 → 6)-β-glucan fraction conjugated by reductive amination to controlled-pore glass beads containing aminopropyl groups. The purified fraction contained a protein of apparent Mr of 70 000 which was radiolabelled by photoaffinity labelling along with twoadditional proteins of 100 000 and 170 000 labelled to a lesser extent. The 70 000 protein represented also the major protein as visualized by silver staining after SDS-PAGE of the purified fraction. Ligand-saturation studies and the kinetics of ligand interaction demonstrated that the hepta-β-glucoside-binding characteristics of the solubilized and enriched protein fractions were very similar to those of the membrane-associated binding sites. The results provide, thus, a clear identification of a membrane protein of 70 000 with high affinity and specificity for a fungal signal molecule capable of triggering plant defence. The results also provide a simple method for the isolation of this protein for further studies at the level of signal perception.

Idioma originalInglés
Páginas (desde-hasta)543-550
Número de páginas8
EstadoPublicada - feb. 1993
Publicado de forma externa


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