, Volume 15, Issue 3, pp 271-279

Gibberellin-photoaffinity labeling of wild oat (Avena fatua L.) aleurone protoplasts


Aleurone protoplasts of wild oat (Avena fatua L.), and subcellular fractions isolated from them, were photoaffinity labeled using the synthetic gibberellin (GA) derivative GA4-17-yl-1′-(1′-thia)propan-3′-ol-4-azido-5-[125I]iodosalicylate. Labeled polypeptides were identified by electrophoresis under denaturing conditions followed by autoradiography. GA-photoaffinity labeling of both intact protoplasts and isolated subcellular fractions led to the covalent attachment of the reagent to many polypeptides. A 50 kD polypeptide in the soluble fraction of homogenates of aleurone protoplasts GA-photoaffinity labeled in vivo showed specific binding. The biologically active GA1, GA4 and GA4-17-yl-1′(1′-thia)propan-3′-ol-4-azidosalicylate completed for binding whereas the biologically inactive GA8 and GA34 did not. The GA-photoaffinity labeling characteristics of this polypeptide suggested that it might interact specifically with biologically active GAs in vivo. Attempts to detect specific GA-binding in in vitro GA-photoaffinity labeling experiments met with only limited success perhaps indicating the labile nature of specific binding observed in vivo. The potential of GA-photoaffinity labeling for identifying GA-binding proteins in aleurone and other GA-responsive tissues is discussed.