Myelin basic protein domains involved in the interaction with actin.
A fluorescence assay was used to measure the interaction of myelin basic protein (MBP) with monomeric actin labeled with a fluorescent compound (IAEDANS). The complex actin-IAEDANS increase the fluorescence in presence of MBP. The enhancement of the fluorescence has a sigmoidal dependence on the concentration of MBP and the fluorescence maximum is reached at a MBP:actin molar ratio of 1:20. The fluorescence maximum in absence of Ca2+ and ATP is 4 times lower than that in their presence 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 define possible interacting domains on MBP involved in the actin-MBP interaction, experiments were performed using MBP-derived peptides obtained under controlled proteolysis of the whole molecule. The fluorescence 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 one near the carboxyl terminus of the MBP molecule. The actin-MBP interaction was also observed using immunoblot and ELISA techniques.
Roth
G A
GA
Departamento de Química Biológica, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Argentina.
Gonzalez
M D
MD
Monferran
C G
CG
De Santis
M L
ML
Cumar
F A
FA
D000199:Actins / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D005456:Fluorescent Dyes; D007106:Immune Sera; D009132:Muscles / Q000378:metabolism; D004676:Myelin Basic Protein / Q000737:chemistry / Q000378:metabolism*; D009282:Naphthalenesulfonates; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D011817:Rabbits; D013050:Spectrometry, Fluorescence; D013116:Spinal Cord / Q000378:metabolism; D016704:Synapsins / Q000378:metabolism
D000199:Actins; D005456:Fluorescent Dyes; D007106:Immune Sera; D004676:Myelin Basic Protein; D009282:Naphthalenesulfonates; D010446:Peptide Fragments; D016704:Synapsins; C006421:1,5-I-AEDANS