Binding of brain spectrin to the 70-kDa neurofilament subunit protein.
Brain spectrin, or fodrin, a major protein of the subaxolemmal cytoskeleton, associates specifically in in vitro assays with the 70-kDa neurofilament subunit (NF-L) and with glial filaments from pig spinal cord. As an initial approach to the identification of the fodrin-binding proteins, a crude preparation of neurofilaments was resolved by electrophoresis on SDS/polyacrylamide gels and then transferred to nitrocellulose paper, which was 'blotted' with 125I-fodrin. A significant binding of fodrin was observed on polypeptides of 70 kDa, 52 kDa and 20 kDa. These polypeptides were further purified and identified respectively as the NF-L subunit of neurofilaments, the glial fibrillary acidic protein (GFP) and the myelin basic protein. The binding of fodrin to NF-L was reversible and concentration-dependent. The ability of the pure NF-L and GFP to form filaments was used to quantify their association with fodrin. a) The binding of fodrin to reassembled NF-L was saturable with a stoichiometry of 1 mol fodrin bound/50 +/- 10 mol NF-L and an apparent dissociation constant Kd = 4.3 x 10(-7) M. b) The binding involved the N-terminal domain of the polypeptide chain derived from the [2-(2-nitrophenylsulfenyl)-3-methyl-3'-bromoindolenine] cleavage of NF-L. c) Binding occurred optimally at physiological pH (6.8-7.2) and salt concentrations (50 mM). d) Interestingly, calmodulin, a Ca2+-binding protein, which has been shown to bind to fodrin, was found to reinforce the binding of fodrin to the NF-L, at Ca2+ physiological concentrations. The binding of fodrin to pure neurofilaments was not affected by the presence of the 200-kDa (NF-H) and the 160-kDa (NF-M) subunits. The apparent dissociation constant for the binding of fodrin to NF-L in the pure NF was 1.0 x 10(-6) M with 1 mol fodrin bound/80 +/- 10 mol NF-L. Moreover, the binding of fodrin to GFP, demonstrated in blot assays, was confirmed by cosedimentation experiments. The apparent dissociation constant Kd for the fodrin binding was 2.8 x 10(-7) M and the maximum binding was 1 mol fodrin/55 +/- 10 mol GFP.
D000818:Animals; D001923:Brain Chemistry; D002118:Calcium / Q000378:metabolism; D002147:Calmodulin / Q000378:metabolism; D002352:Carrier Proteins / Q000378:metabolism*; D005904:Glial Fibrillary Acidic Protein / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007381:Intermediate Filament Proteins / Q000378:metabolism*; D008840:Microfilament Proteins / Q000378:metabolism*; D008970:Molecular Weight; D016900:Neurofilament Proteins; D009994:Osmolar Concentration; D013552:Swine; D013049:Spectrin
D002118:Calcium; D002147:Calmodulin; D002352:Carrier Proteins; D005904:Glial Fibrillary Acidic Protein; D007381:Intermediate Filament Proteins; D008840:Microfilament Proteins; D016900:Neurofilament Proteins; D013049:Spectrin