Purification and photoaffinity labeling of fusicoccin receptors from maize.
Crude soluble proteins from plasma membranes of maize shoots were purified (following the increase of fusicoccin-binding specificity) by using an original multi-step HPLC procedure. The method, based on a combination of adsorption, ion-exchange and gel-filtration chromatographies, is quick, efficient and does not damage the binding activity. It allows a 5000-fold increase of specific activity; SDS/PAGE of purified fractions shows two doublets that correspond to proteins with apparent molecular masses of 90 kDa and 30 kDa. Crude or partially purified material was irradiated for various periods in the presence of a tritiated azido analogue of fusicoccin. The electrophoretic analysis of the irradiated material shows that with a short irradiation time only the 90-kDa band is radiolabeled, whereas, as the irradiation time increases, a 30-kDa band becomes radiolabeled and less radioactivity is detected in the 90-kDa band. Irradiation of the crude material in the absence of the analogue results in a decrease of the binding capability of fusicoccin. The irradiated preparation also shows a decrease of photolabeling of the 90-kDa band. Our data suggest that the 90-kDa protein is the functional fusicoccin receptor. This conclusion is at variance with results of other authors who suggest the 30-kDa protein as the true receptor.
Department of Biology, University of Rome Tor Vergata, Italy.
P
P
Aducci
A
A
Ballio
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V
Fogliano
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MR
Fullone
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Marra
N
N
Proietti
D001665:Binding Sites; D002462:Cell Membrane / Q000378:metabolism; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D006027:Glycosides / Q000378:metabolism; D010940:Plant Proteins; D011869:Radioligand Assay; D011956:Receptors, Cell Surface / Q000302:isolation & purification* / Q000378:metabolism; D003313:Zea mays / Q000737:chemistry*
D006027:Glycosides; D010940:Plant Proteins; D011956:Receptors, Cell Surface; C080425:fusicoccin receptor protein, plant; C007808:fusicoccin