P-4 電子顕微鏡観察プローブ(TEMプローブ)を利用した就眠運動関連受容体局在性のナノスケール直接観察(ポスター発表の部)

Potassium isolespedezate (1) is a leaf-opening substance of leguminous plant, Cassia mimosoides L. We already revealed that the target cell of 1 is a motor cell, but exact localization of the receptor protein in motor cell is still unknown. Therefore. we employed transmission electron microscope (TEM) to observe the localization of the receptor protein. We developed TEM probe 4 that introduced benzophenone as photoaffinity unit. and FITC as detection unit, to 1. Receptor covalently binds with 4 by photoaffinitylabeling. And by following nano-gold binding anti-FITC antibody treatment, receptor is labeled by nano-gold. This nano-gold can be observed by electron microscope. In making ultrathin section for electron microscopy, photolabeling and antibody treatment was produced before fixation. Dehydration, and embedding to avoid denaturation of the receptor and degradation of the antigen. But in this procedure. normal anti-FITC antibody was too large to penetrate into the thick plant section. So we improved permeability of antibody by using nano-gold binding anti-FITC antibody constituted of 1.4nm diameter gold cluster and Fab fragment of antibody. Photoaffinitylabeling of plant section using 4 and following treatment by nano-gold binding anti-FITC antibody gave TEM image that the receptor labeled by nano-gold particle is localized on the plasma membrane. Then. we revealed that the receptor protein of 200kDa concerning nyctinasty is localized on the plasma membrane. This result gave an important clue for the molecular mechanism of nyctinasty.