SPECIFIC PHOTOAFFINITY-LABELING OF A THYLAKOID MEMBRANE-PROTEIN WITH AN AZIDO-CYTOKININ AGONIST

A cytokinin-binding peptide (CBP) of 46 kDa (Thy46) has been identifie d in thylakoid membranes of pea chloroplasts, by photoaffinity labelli ng with tritiated 1-(2-azido-6-chloropyrid-4-yl)-3-phenylurea ([H-3]az idoCPPU), a urea-type cytokinin agonist. The labelled peptide is also detected in Nicotiana plumbaginifolia, Nicotiana tabacum and spinach t hylakoid membranes, but is absent in thylakoid membranes of Chlamydomo nas reinhardtii. A pharmacological study of the interaction of this pe ptide with different cytokinin agonist molecules has been achieved. Ur ea derivatives are the most efficient competitors of photolabelling, a nd this efficiency is in good agreement with the cytokinin activity of these compounds. A quantitative analysis of the displacement of the p hotoaffinity labelling of the peptide by increasing concentrations of CPPU indicates an apparent dissociation constant of 1 mu M for this li gand. Purine-type cytokinins are weaker competitors than urea-type mol ecules, but the efficiency of the competition is also correlated to th eir respective cytokinin activity. A partial purification of Thy46 by a protocol involving ion exchange chromatography and 2D-gel electropho resis is described.