|Budget Amount *help
¥2,000,000 (Direct Cost : ¥2,000,000)
Fiscal Year 1994 : ¥900,000 (Direct Cost : ¥900,000)
Fiscal Year 1993 : ¥1,100,000 (Direct Cost : ¥1,100,000)
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.