REGIOSELECTIVE BINDING OF 2,5-HEXANEDIONE TO HIGH-MOLECULAR-WEIGHT RAT NEUROFILAMENT PROTEINS IN-VITRO

Previous studies have shown selective binding of the neurotoxicant 2,5 -hexanedione (2,5-HD) to carboxyl-terminal domains of rat neurofilamen t (NF) M and H proteins in vitro. The present study was designed to fu rther localize this binding in native rat NF preparations exposed to [ C-14]2,5-HD. Purified M and H proteins from the 2,5-HD-treated NFs wer e subjected to cyanogen bromide (CNBr) cleavage, and the resultant pep tides were separated by tris-tricine SDS-PAGE and electroblotted to PV DF membranes. Peptides were identified by direct sequencing of stained bands and the relative radiolabeling of each peptide was determined b y comparing band intensities in fluorographed blots. For NF-M, the hig hest label was found in CNBr 10, a peptide corresponding to residues 6 78-846 at the extreme carboxyl terminus. This region of the protein in cludes three highly conserved lysine-containing sequences believed to be critical to its function. For NF-H, the greatest binding was locali zed in CNBr7+8, representing an incomplete cleavage product of residue s 390-810. This peptide contains essentially all of the phosphorylatio n sites in the carboxyl terminus of NF-H, a domain believed to control NF interactions in the axon. Only minor radiolabeling was observed in other M or H peptides. Extensive dephosphorylation of NFs prior to 2, 5-HD exposure had no effect on relative adduct levels in each protein. These results provide additional support for limited and specific bin ding of 2,5-HD to neurofilaments and indicate that the phosphorylation stat of the protein may not substantially influence this binding.