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 neurofilament (NF) M and H proteins in vitro. The present study was designed to further localize this binding in native rat NF preparations exposed to [14C]2,5-HD. Purified M and H proteins from 2,5-HD-treated NFs were subjected to cyanogen bromide (CNBr) cleavage, and the resultant peptides were separated by Tris-tricine SDS-PAGE and electroblotted to PVDF membranes. Peptides were identified by direct sequencing of stained bands and the relative radiolabeling of each peptide was determined by comparing band intensities in fluorographed blots. For NF-M, the highest label was found in CNBr 10, a peptide corresponding to residues 678-846 at the extreme carboxyl terminus. This region of the protein includes three highly conserved lysine-containing sequences believed to be critical to its function. For NF-H, the greatest binding was localized in CNBr 7 + 8, representing an incomplete cleavage product of residues 390-810. This peptide contains essentially all of the phosphorylation 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 binding of 2,5-HD to neurofilaments and indicate that the phosphorylation state of the protein may not substantially influence this binding.
DeCaprio
A P
AP
Wadsworth Center for Laboratories and Research, New York State Department of Health, Albany 12201, USA.
Kinney
E A
EA
Fowke
J H
JH
D000818:Animals; D001667:Binding, Competitive; D002250:Carbon Radioisotopes; D002800:Cholinesterase Inhibitors / Q000378:metabolism* / Q000633:toxicity; D003488:Cyanogen Bromide / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006588:Hexanones / Q000378:metabolism* / Q000633:toxicity; D066298:In Vitro Techniques; D007553:Isotope Labeling; D008297:Male; D008970:Molecular Weight; D016900:Neurofilament Proteins / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D009498:Neurotoxins / Q000378:metabolism* / Q000633:toxicity; D010766:Phosphorylation; D051381:Rats; D017208:Rats, Wistar; D013116:Spinal Cord / Q000187:drug effects / Q000378:metabolism
D002250:Carbon Radioisotopes; D002800:Cholinesterase Inhibitors; D006588:Hexanones; D016900:Neurofilament Proteins; D009498:Neurotoxins; C051774:neurofilament protein H; C061195:neurofilament protein M; C011269:2,5-hexanedione; D003488:Cyanogen Bromide