Interaction of annexins with membrane skeletal proteins in brain

膜联蛋白与大脑中膜骨骼蛋白的相互作用

基本信息

批准号：
05680676
负责人：
INUI Makoto
金额：
$ 1.28万
依托单位：
Osaka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1993
资助国家：
日本
起止时间：
1993 至 1994
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-05680676/
关键词：
Membrane skeleton Calcium regulation Annexins Calspectin SynapsinI Phospholipids 燐脂質

项目摘要

To elucidate the physiological significance of annexin VI in brain, we developed a method to detect annexin VI-binding proteins using ^<125>I-annexin VI on a nitrocellulose sheet to which rat brain proteins were electrophoretically transferred after SDS polyacrylamide gel electrophoresis. When we anlayzed the wohole homogenate of rat brain cortex, significant binding of annexin VI was observed at the protein bands of Mr450K,350K,240K,180K,120-140K,105K,80K,63K,48K,32K,27K,23K and 16K.The binding was dependent on Ca^<2+> and phosphatidylserine (PS) or phosphatidic acid. No significant binding was observed with phosphatidylcholine, phosphatidylethanolamine, or phosphatidylinositol.A line of evidences indicated that the binding is a direct protein-protein interaction between annexin VI and its binding proteins. Of annexin VI-binding proteins, the 80K and the 240K proteins were identified to be synapsin I and calspectin, respectively. The bindings of annexin VI to these proteins were also observed in the native state. Annexin VI bound to the NH2-terminal head region of synapsin I.The binding was inhibited by phosphorylation of synapsin I by cAMP-PK or by CaM-PK II.Annexin VI inhibited the interaction between calspectin and Factin by binding to calspectin in the presence of Ca^<2+> and PS,indicating a regulatory role of annexin VI in the modulation of membrane skeleton by Ca^<2+>. Thus, the methods used here were proven to be quite powerful in identifying the molecular targets of annexin VI.The physiological roles of annexin VI in brain can be elucidated by examining the effects of annexin VI on its targets.

为了阐明与大脑中膜联蛋白VI的生理意义，我们开发了一种方法，在硝基纤维素片上使用 ^<125> i- annexin VI检测膜联蛋白VI结合蛋白，大鼠脑蛋白是在SDS PolyAcrylamide Gel电子电泳后将大鼠脑蛋白转移到的，该蛋白是电的转移。当我们对大鼠脑皮层的wohole匀浆缠结时，在MR450K，350K，240K，180K，180K，120-140K，105K，80K，80K，63K，63K，48K，48K，32K，27K，27K，23K和16K上观察到膜联蛋白VI的显着结合。磷脂酸。没有观察到磷脂酰胆碱，磷脂酰乙醇胺或磷脂酰肌醇的明显结合。证据线表明结合是膜联蛋白VI及其结合蛋白之间的直接蛋白质 - 蛋白质相互作用。在膜联蛋白VI结合蛋白中，分别鉴定出80K和240K蛋白是突触素I和Calspectin。在本地状态也观察到了膜联蛋白VI与这些蛋白质的结合。与NH2-末端末端区域结合突触的膜联蛋白VI。结合通过CAMP-PK或CAM-PK II.Annexin vi抑制突触I的磷酸化抑制了Calspectin和factin与Calspectin在CA^<2+>和PS的作用中的作用，通过与CALSPECTIN结合，抑制了Calspectin和factin之间的相互作用，从而抑制了结合。 Ca^<2+>。因此，此处使用的方法在识别膜联蛋白VI的分子靶标方面非常有力。可以通过检查膜联蛋白VI对靶靶标的影响来阐明脑周期蛋白VI在大脑中的生理作用。