Detection of membrane-bound cytokinin-binding proteins in Arabidopsis thaliana cells

In order to isolate cytokinin-binding proteins (CBPs), we have developed ne w affinity probes: constituted of a cytokinin such as zeatin riboside ([9R] Z) conjugated to a carrier protein. These probes were used for detecting CB Ps in an ELISA procedure. The efficiency of the cytokinin conjugate in dete cting CBPs was controlled with protein model: proteins having an affinity f or cytokinin such as the monoclonal anti-[9R]Z antibodies did bind the cyto kinin conjugate whereas proteins unable to bind cytokinin such as bovine se rum albumin did not. Using these new affinity probes, we showed that CBPs a re present in the membrane fraction of in vitro cultured Arabidopsis thalia na cells. The nature of the protein at the detected binding sites was demon strated by submitting the microsomal proteins to a proteolytic treatment, w hich was found to eradicate the binding. Free biologically active cytokinin s or monoclonal anti-[9R]Z antibodies inhibited the binding, thus showing t he specificity of the interaction. The detected CBPs were partially solubil ized from the membranes with potassium chloride, indicating their periphera l membrane location. The separation by anion exchange chromatography of sol ubilized microsomal proteins revealed the existence of two different CBPs. They were present at higher levels in cells during the exponential growth p hase.