PURIFICATION AND PHOTOAFFINITY-LABELING OF FUSICOCCIN RECEPTORS FROM MAIZE

Crude soluble proteins from plasma membranes of maize shoots were puri fied (following the increase of fusicoccin-binding specificity) by usi ng an original multi-step HPLC procedure. The method, based on a combi nation of adsorption, ion-exchange and gel-filtration chromatographies , is quick, efficient and does not damage the binding activity. It all ows a 5000-fold increase of specific activity; SDS/PAGE of purified fr actions shows two doublets that correspond to proteins with apparent m olecular masses of 90 kDa and 30 kDa. Crude or partially purified mate rial was irradiated for various periods in the presence of a tritiated azido analogue of fusicoccin. The electrophoretic analysis of the irr adiated 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 o f the analogue results in a decrease of the binding capability of fusi coccin. The irradiated preparation also shows a decrease of photolabel ing of the 90-kDa band. Our data suggest that the 90-kDa protein is th e functional fusicoccin receptor. This conclusion is at variance with results of other authors who suggest the 30-kDa protein as the true re ceptor.