Five proteins having molecular masses of 90, 67, 37, 36, and 32 kDa (p90, p67, p37, p36, and p32, respectively) were identified in the particulate fractions of pig brain cortex and pig spinal cord prepared in the presence of 0.2 mM Ca2+ and further purified using a protocol previously described for the purification of calpactins. Proteins p90, p37, and p36 are related to annexins I and II. Annexin II, represented by p90, is found as an heterotetramer, composed of two heavy chains of 36 kDa and two light chains of 11 kDa, and as a monomer of 36 kDa. Protein p37, which differs immunologically from p36, is a monomer and could be related to annexin I. All three proteins are Ca(2+)-dependent phospholipid- and F-actin-binding proteins; they are phosphorylated on a serine and on a tyrosine residue by protein kinases associated with synaptic plasma membranes. Purified p36 monomer and p36 heterotetramer proteins bind to actin at millimolar Ca2+ concentrations. The stoichiometry of p36 binding to F-actin at saturation is 1:2, corresponding to one tetramer or monomer of calpactin for two actin monomers (KD, 3 x 10(-6) M). Synaptic plasma membranes supplemented with the monomeric or tetrameric forms of p36 phosphorylate the proteins on a serine residue. The monomer is phosphorylated on a serine residue by a Ca(2+)-independent protein kinase, whereas the heterotetramer is phosphorylated on a serine residue and a tyrosine residue by Ca(2+)-dependent protein kinases. Antibodies to brain p37 and p36 together with antibodies to lymphocytes lipocortins 1 and 2 were used to follow the distribution of these proteins in nervous tissues. Polypeptides of 37, 34, and 36 kDa cross-react with these antibodies. Anti-p37 and antilipocortin 1 cross-react on the same 37- and 34-kDa polypeptides; anti-p36 and antilipocortin 2 cross-react only on the 36-kDa polypeptides.

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Brain spectrin, or fodrin, a major protein of the subaxolemmal cytoskeleton, associates specifically in in v i m 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 1251-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 -t 10 mol NF-L and an apparent dissociation constant Kd = 4.3 x lo-' 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 Caz+-binding protein, which has been shown to bind to fodrin, was found to reinforce the binding of fodrin to the NF-L, at Ca2 Brain spectrin of fodrin is a major protein in neurons, where it represents 3% of the membrane protein [l]. By immunofluorescence microscopy, fodrin has been localized and found associated to the plasmalemma in cell bodies, dendrites, axons and nerve terminals [2, 31. This association appears to involve two membrane-attachment proteins, ankyrin and synapsin I. They have been recently purified from brain [4, 51 and shown to possess the same functional properties as those described for ankyrin and protein 4.1 in erythrocytes: their presence suggests that, like erythrocyte spectrin, fodrin may play an active role in stabilizing the membrane of neurons as well as in immobilizing some integral membrane proteins as receptors, which are yet to be characterized.Unlike erythrocyte spectrin, fodrin is mobile and undergoes axonal transport. Axonal transport studies [6] have shown that fodrin is the only known protein to migrate at different rates along the axon, with vesicles, organelles and mitochondria (50 mm/day), with actin and myosin (5 mm/ day) and with microtubules and neurofilaments (0.5 -I mm/ Correspondence to L. A. Pradel, Institut de Biologie PhysicoChimique, 13 Rue Pierre el Marie Curie, F-75005 Paris, FranceAbbreviations. NF-L, NF-M, NF-H, 70-kDa, 160-kDa, 200-kDa neurofilament subunits; GFP, glial filament protein; BNPS-skatole, 2-(2-nitrophenylsulfenyl)-3-methyl-3'-bromoindolenine; PMSF, phenylmethylsulphonylfluoride. day). These various rates of migration can be explained if fodrin, a multifunctional protein, is capable of...

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