Photoaffinity labeling and partial purification of the putative plant receptor for the fungal wilt-inducing toxin, fusicoccin
✍ Scribed by Martin Feyerabend; Elmar W. Weiler
- Book ID
- 104752724
- Publisher
- Springer-Verlag
- Year
- 1989
- Tongue
- English
- Weight
- 885 KB
- Volume
- 178
- Category
- Article
- ISSN
- 0032-0935
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✦ Synopsis
The high-affinity fusicoccin-binding protein (FCBP) was solubilized from plasma-membrane vesicles prepared from leaves of Vicia faba L. by aqueous two-phase partitioning. Conditions for the solubilization of intact FCBP-radioligand complexes were worked out. About 60-70% of the complexes can be solubilized with 50-60 mM nonanoyl-N-methylglucamide in the presence of I mgml-1 soybean phosphatidylcholine, type IV S, and 20% (v/v) glycerol at pH 5.5. The slow dissociation of the radioligand, 9'-nor-fusicoccin-8'-alcohol-[3H] from the FCBP at low temperatures permits the purification of FCBP-radioligand complexes at 4-10 ~ C by fast protein liquid chromatography on anion-exchange and gel permeation columns. The FCBP, extracted from plasma membranes with cholate and chromatographed in the presence of this detergent, gave an apparent molecular mass (Mr) of 80___20 kDa on gel permeation columns under the conditions used. By comparison of the elution profiles of the fraction most enriched in FCBP-radioligand complexes with polypeptide patterns obtained on sodium dodecyl sulfate-polyacrylamide gels, a polypeptide with an Mr of approx. 34 kDa co-separated with the radioactivity profile. A second, faint band of approx. 31 kDa was sometimes also observed co-electrophoresing. Photoaffinity labeling of plasma-membrane vesi-