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 × 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 × 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 × 10−7 M and the maximum binding was 1 mol fodrin/55 ± 10 mol GFP.