| Co-Investigator(Kenkyū-buntansha) |
MASUDA Yayoi Jichi Med. Sch., Dept. of Anat., Research associate, 医学部, 助手 (20254906)
YAMAKADO Makoto Jichi Med. Sch., Dept. of Anat., Assistant Professor, 医学部, 助教授 (80010114)
|
|---|
| Research Abstract |
The activation of transducin (GTP binding protein) occurs, shortly after photon reception by rhodopsin, and this activated tranducin further activates 3', 5' -cyclic nucleotide phosphodiesterase (PDE) produces rapid decrese of cGPM concentration in the cytoplasm of visual cells. The low cGPM concentration closes the ion channel on the surface of plasma membrane and results to induce action potential by hyper polarization. The aim of this project was to visualize these dynamic processes, directory on the ultastructure by cytochemistry. It has become feasible to investigate the mechanism of rapid enzymatic activation in visual cells, by the developing rapid freeze substitution enzyme cytochemistry in my laboratory. The method visualizes cnzyme locations after atopping all molecular movement within a few milli-seconds at liquid nitrogen or helium temperature. To visualize precise enzyme sites, it is also essentially important to guarantee the fine localization of cytochemical reaction product. On this view point the question was on the PDE method, because the action of protease and lipase contaminated in the snake venom, which was added in the reaction medium to mediate the second step, by which GPM undergoes breakdown to guanosine and phoshate, give destructive damage on the tissue. The liberated phosphate was captured by lead nitrate as precipitate of lead phosphate. Therefore, we have tried to replace the snake venom with purified 5' -nucleotidase (5' -N) to improve ultrastructure and localization of reaction product. The localization site of 3', 5' -PDE activity was also compared with 2', 3' -PDE.Another improvement of 5' -N, the terminal step of cGPM cascade, was also tried, changing capture agent from lead nitrate to lead citrate which enables us to react at pH 8.0. With this new method, the positive reaction was demonstrated sharply from pigment epithelinm to outhr of the retina, where visual cells extend.
|
