Detection of 210 kDa receptor protein for a leaf-movement factor by using novel photoaffinity probes

A Target Protein for Potassium Isolespedezate, A Bioactive Metabolite Controlling Nyctinasty

“…Hydrophilic 1 clearly cannot penetrate the plasma membrane to bind with CTPL, indicating an involvement of a membrane transporter to carry 1 into the cytosol. Previously, we reported that 1 interacts with a 210-kDa membrane protein of the motor cell using a biotin-conjugated photoaffinity probe (6) 18 . This result suggested that the large size of probe 6 would lower the efficiency of membrane trafficking via a transporter.…”

Interplay between the Chemical Space and the Biological Space

“…The three most common photophores used in target identification studies are arylazides, benzophenones, and diazirines (Figure 3.15) [117]. Fujii et al used biotinylated photocrosslinking probes to discover receptors for the leaf-movement factor potassium lespedezate, a ligand whose concentration governs circadian rhythmic leaf-movement in leguminous plants (Figure 3.16) [118]. Three different probes were synthesized in order to examine the relationship between bioactivity and photocrosslinking efficiency of the probes, as systematic studies of such relationships are lacking.…”

Endogenous factors involved in the regulation of movement and "memory" in plants

“…We synthesized a biologically active biotinylated photoaffinity [33][34][35][36] probe 15 based on potassium isolespedezate 16, a leafopening factor of Cassia mimosoides [37,38]. The photoaffinity probe 15 was effective at concentrations of 1 × 10 -4 M in a bioassay with leaves of C. mimosoides, and had an activity that was one-hundredth as effective as the natural product.…”

“…After cross-linking, the membrane fraction was analyzed by SDS-PAGE and Western blotting was performed. Two probe-coupled target proteins of 210 and 180 kDa were detected by chemiluminescence [37,38]. Specific binding of the probe was confirmed by the disappearance of the corresponding bands during photolabeling in the presence of an excess amount of nonlabeled leaf-opening substance.…”