The goal of this application is to continue our research on the biochemistry and assembly of mammalian neurofilaments. Specifically, we propose to characterize a newly discovered 66 kD protein that is a novel subunit of neurofilament. Its relationship with the known triplet proteins will be explored in reassembly. We will isolate the 66 kD neurofilament protein by FPLC chromatography, a technique we recently established. the soluble pool of this protein will be defined and the possible mechanism such as phosphorylation, that leads to the solubilization of this protein will be evaluated. Antisera specific to the 66 kD protein that do not crossreact with any of the neurofilament triplets have been prepared and they microscopy and in EM to localized this protein. The developmental expression of the 66 kD protein in both CNS and PNS will be documented. The distribution of the 66 kD protein will be mapped by both immunocytochemical techniques and quantitative immunoblottings. We will use the STEM facility in Brookhaven National Laboratory 66 kD protein. The effect of phosphorylation on the disassembly of the 66 kD protein and the 68 kD neurofilament protein (NF-l) will be investigated. We will measure reassembly of iodinated neurofilament proteins to proteins that are bound to nitrocellulose sheets.
