Neurofilaments are intermediate filaments of neurons, and comprise the major cytoskeletal structure of large neuritic processes. Alterations of neurofilament structure have been postulated as a major feature of Alzheimer's disease (presenile and senile dementia) and of aging brains, and have been found in other neuropathic conditions. Neurofilaments are comprised of three distinct polypeptide chains of 68,000 (P68), 150,000 (P150) and 200,000 (P200) molecular weight. We propose to isolate large quantities of the constituent subunits for structural analysis, in order to obtain fundamental information about the nature of the individual subunits, their interactions and interrelationships. Insight into their mode of association and assembly will also be gained. Physico-chemical analyses will be used to determine whether or not there are discrete association states of the three neurofilament subunits. Sequence analysis of the purified subunits will be performed. Peptide maps will be obtained using specific cleavage agents, and separated by ion-exchange and hydrophobic interaction chromatography (HPLC) and gel filtration. These maps will be used to compare possible common sequences between the three subunits, to identify peptides exposed on the surface of intact neurofilaments, and to isolate peptides which are phosphorylated in the native neurofilaments. both poly- and mono-clonal antibodies will be used as probes of cross-reaction between the subunits and of surface topography of the filaments. Chemical modification of intact neurofilaments will also be used to map exposed residues.
