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.

为了阐明膜联蛋白VI在脑中的生理意义，我们开发了一种使用硝化纤维素片上的^ 125 I-膜联蛋白VI来检测膜联蛋白VI结合蛋白的方法，在SDS聚丙烯酰胺凝胶电泳后将大鼠脑蛋白电泳转移到硝化纤维素片上。对大鼠大脑皮质全匀浆进行分析，Mr450K、350K、240K、180K、120-140K、105K、80K、63K、48K、32K、27K、23K、16K蛋白条带均观察到annexin VI显着结合。 .结合依赖于Ca 2+ 和磷脂酰丝氨酸(PS) 或磷脂酸。没有观察到与磷脂酰胆碱、磷脂酰乙醇胺或磷脂酰肌醇的显着结合。一系列证据表明该结合是膜联蛋白VI与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，80K 和 240K 蛋白分别被鉴定为突触蛋白 I 和 calspectin。在天然状态下也观察到膜联蛋白 VI 与这些蛋白质的结合。膜联蛋白 VI 与突触蛋白 I 的 NH2 末端头部区域结合。突触蛋白 I 的磷酸化被 cAMP-PK 或 CaM-PK II 抑制。膜联蛋白 VI 通过在存在的情况下与钙调蛋白结合，抑制钙调蛋白与 Factin 之间的相互作用Ca^<2+> 和 PS，表明膜联蛋白 VI 在 Ca^<2+> 调节膜骨架中的调节作用。因此，这里使用的方法被证明在识别膜联蛋白VI的分子靶标方面非常有效。通过检查膜联蛋白VI对其靶标的影响，可以阐明膜联蛋白VI在大脑中的生理作用。