Interaction of annexins with membrane skeletal proteins in brain

为了阐明膜联蛋白VI在脑内的生理意义，我们建立了一种利用硝酸纤维素膜上的膜联蛋白VI检测膜联蛋白VI结合蛋白的方法，大鼠脑蛋白质经SDS-聚丙烯酰胺凝胶电泳后被转移到硝酸纤维素膜上。当我们分析大鼠大脑皮层腔匀浆时，发现Annexin VI在Mr450K，350K，240K，180K，120-140K，105K，80K，63K，48K，32K，27K，23K和16K的蛋白区带上有明显的结合，结合依赖于Ca~(2+)和磷脂酰丝氨酸(PS)或磷脂酸。未观察到磷脂酰胆碱、磷脂酰乙醇胺或磷脂酰肌醇的结合。一系列证据表明，这种结合是膜联蛋白VI与其结合蛋白之间的直接蛋白质相互作用。在膜联蛋白VI结合蛋白中，80K和240K蛋白分别被鉴定为突触素I和钙调素。在天然状态下，也观察到Annexin VI与这些蛋白的结合。Annexin VI与突触素I的NH2末端头部区域结合，cAMP-PK或CaM-PK II对突触素I的磷酸化抑制作用。Annexin VI在钙离子和PS存在的情况下，通过与Calspectin结合来抑制Calspectin和Factin之间的相互作用，表明Annexin VI在Ca^&lt；2+和PS对膜骨架的调控中起着调节作用。因此，本文所用的方法被证明在确定膜联蛋白VI的分子靶点方面是相当有效的。通过检测膜联蛋白VI对其靶点的影响，可以阐明膜联蛋白VI在脑中的生理作用。

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.