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Study of phototransduction micromachinary on the disk membrane

圆盘膜上光转导微机械的研究

基本信息

批准号：
14570009
负责人：
NISHIZAWA Yuji
金额：
$ 2.24万
依托单位：
Nagoya University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2002
资助国家：
日本
起止时间：
2002 至 2003
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14570009/
关键词：
photoreceptor retina immunocytochemistry micromachinary mass spectrometry ROS tau tubulin

项目摘要

The process of outer segment disk formation occurs throughout the life of an organism, providing a mechanism for continual renewal of outer segment components. On a disk membrane with dynamic structure, a lot of proteins related to phototransduction, such as rhodopsin, exist and the transduction cascade works when the segment has received light. Since the cascade reaction progresses very quickly, formation of micromachinery that accumulates the proteins can be predicted. Immunocytochemistry revealed that tubulin alpha, beta, gamma and tau were co-localized on the disk membrane. To identify the tubulin specific binding proteins in outer segments, we demonstrated immunoprecipitation and mass spectrometry. Some proteins were identified by mass spectrometry. The tau and an annexin II were co-precipitated with tubulin beta and gamma. These results suggest that the tubulin plays the role of the scaffold of a phototransduction micromachinary. Since the bovine retina stopped obtaining for the BSE of the bovine, we changed material into the frog retina. We prepared frog ROS fraction and analyzed the fraction by 2D-PAGE and mass spectrometry. Thirty five proteins had been identified when 136-gel spots were analyzed. Forteen proteins were rhodopsin, The proteins of 14 originated in outer segment and contained rhodopsin, phosphodiesterase alpha and beta, transducin alpha and beta, arrestin and s-modulin. The proteins of 18 originated in inner segment and contained HSC70,enolase, GTP-rho binding protein, neuronal intermediate filament and ATP synthase. The actin, and a tubulin alpha and beta were common to both.

外节段盘的形成过程发生在生物体的整个生命过程中，为外节段成分的持续更新提供了一种机制。在具有动态结构的圆盘膜上，存在许多与光转导相关的蛋白质，如视紫红质，当片段受光时，转导级联作用。由于级联反应进展非常快，可以预测积累蛋白质的微机械的形成。免疫细胞化学显示微管蛋白α、β、γ和tau共定位于盘膜上。为了鉴定外节段中的微管蛋白特异性结合蛋白，我们进行了免疫沉淀和质谱分析。对部分蛋白质进行了质谱学鉴定。Tau和膜联蛋白II与微管蛋白β和伽马蛋白共沉淀。这些结果表明，微管蛋白起到了光转导微机械支架的作用。由于牛的视网膜停止获取牛的疯牛病，我们把材料换成了青蛙的视网膜。我们制备了蛙类ROS组分，并用2D-PAGE和MS对其进行了分析。对136个凝胶斑点进行分析，共鉴定出35个蛋白质。14种蛋白质为视紫红质，其中14种蛋白质起源于外段，包括视紫红质、磷酸二酯酶α和β、转导蛋白α和β、芳香素和S-调制素。其中18个蛋白起源于内段，包括HSC70、烯醇化酶、GTP-Rho结合蛋白、神经元中间丝和ATP合成酶。肌动蛋白和微管蛋白α和β都是共同的。