Abstract
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 1 mg· ml-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. 34kDa 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 vesicles with the new compound 9′-nor-8′[(3,5-[3H]-4-azidobenzoy)ethylenediamine]-fusicoccin ([3H]ABE-FC) and subsequent separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis labeled a single band with an Mr of 35±1 kDa. Labeling in this band was strongly reduced when the membranes were incubated with [3H]ABE-FC in the presence of 0.1–1 μM fusicoccin. From our data, we conclude (i) that the 34-35-kDa polypeptide represents the FCBP and (ii) that in detergent extracts of plasma membranes this polypeptide is probably present as a di- or trimeric structure.
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Abbreviations
- ABE-FC:
-
[(4-azidobenzoyl)-ethylenediamine]-fusicoccin
- ABE-NHS:
-
(4-azidobenzoyl)-N-hydroxysuccinimide ester
- FC:
-
fusicoccin
- FCBP:
-
fusicoccin-binding protein
- FCol:
-
9′-norfusicoccin-8′-alcohol
- MAB:
-
monoclonal antibody
- Mega-9(10):
-
nonanoyl(decanoyl)-N-methylglucamide
- Mr :
-
apparent molecular mass
- PMSF:
-
phenylmethyl-sulfonyl fluoride
- SDS-PAGE:
-
sodium dodecyl sulfate-polyacrylamide gel electrophoresis
- TCA:
-
trichloroacetic acid
- Tris:
-
2-amino-2-(hydroxymethyl)-1,3-propanediol
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Feyerabend, M., Weiler, E.W. Photoaffinity labeling and partial purification of the putative plant receptor for the fungal wilt-inducing toxin, fusicoccin. Planta 178, 282–290 (1989). https://doi.org/10.1007/BF00391855
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DOI: https://doi.org/10.1007/BF00391855