MYELIN BASIC-PROTEIN DOMAINS INVOLVED IN THE INTERACTION WITH ACTIN

A fluorescence assay was used to measure the interaction of myelin bas ic protein (MBP) with monomeric actin labeled with a fluorescent compo und (IAEDANS). The complex actin-IAEDANS increase the fluorescence in presence of MBP. The enhancement of the fluorescence has a sigmoidal d ependence on the concentration of MBP and the fluorescence maximum is reached at a MBP: actin molar ratio of 1 : 20. The fluorescence maximu m in absence of Ca2+ and ATP is 4 times lower than that in their prese nce although it is reached at the same MBP: actin molar ratio. Similar behavior is observed when synapsin replaces MBP, while acetylated MBP and bovine serum albumin fail to induce any fluorescence change. To d efine possible interacting domains on MBP involved in the actin-MBP in teraction, experiments were performed using MBP-derived peptides obtai ned under controlled proteolysis of the whole molecule. The fluorescen ce changes induced by the different peptides depend on their location in the native protein and can not be explained simply by a difference in the net charge of the peptides. The results suggest that two sites are involved in the interaction. A Ca2+/ATP-dependent site located in the amino-terminal region (peptide 1-44) and a Ca2+/ATP-independent on e near the carboxyl terminus of the MBP molecule. The actin MBP intera ction was also observed using immunoblot and ELISA techniques.