Limited and selective adduction of carboxyl-terminal lysines in the high molecular weight neurofilament proteins by 2,5-hexanedione in vitro.
2,5-Hexanedione (2,5-HD) induces a toxic neuropathy characterized by massive, focal axonal neurofilament (NF) accumulation. Covalent interaction of 2,5-HD with NF protein amines, resulting in pyrrole adduct formation, has been proposed as a critical step in its mechanism. The present study was undertaken to evaluate the hypothesis of selective 2,5-HD/lysine modification, by quantitating in vitro adduction in the NF proteins and in specific polypeptide domains of each protein. Native rat spinal cord NFs were exposed to 0-212.5 mM [14C]2,5-HD for 2-16 h (37 degrees C under argon), followed by removal of non-covalently bound radioactivity. Incorporation of radioactivity and pyrrole formation in NFs increased linearly with 2,5-HD concentration and biphasically with time. SDS-PAGE and fluorography demonstrated prominent labeling of the three NF subunit proteins (H, M, and L), in addition to high-MW, crosslinked material derived from NF-H and -M. Mild chymotryptic cleavage was employed to isolate the carboxyl-terminal 'tail' domains of NF-H and -M, and the pooled amino-terminal NF 'rod' regions, all of which were radiolabeled. Specific activity (mol adduct/mol protein) of adducted NF proteins and polypeptide domains was determined by scintillation counting of electroeluted proteins. Stable binding in the NF-H and -M proteins was 4- to 6-fold higher than in the NF-L protein at all 2,5-HD concentrations, with specific activities of approximately 6.9, 4.7, and 1.3 mol/mol protein, respectively, at 212.5 mM. Approximately 70-80% of NF-H and -M binding was localized to the tail domains. In contrast, NF-L and pooled rod domain adduction did not substantially exceed 1 mol/mol protein. These findings provide the first direct evidence for limited and selective pyrrole adduction in the NF proteins following 2,5-HD exposure.
DeCaprio
A P
AP
Wadsworth Center for Laboratories and Research, New York State Department of Health, Albany.
Fowke
J H
JH
D000818:Animals; D001665:Binding Sites; D002250:Carbon Radioisotopes; D004591:Electrophoresis, Polyacrylamide Gel; D006588:Hexanones / Q000378:metabolism*; D007700:Kinetics; D008239:Lysine; D046911:Macromolecular Substances; D008854:Microscopy, Electron; D016900:Neurofilament Proteins / Q000302:isolation & purification / Q000378:metabolism* / Q000648:ultrastructure; D009498:Neurotoxins / Q000378:metabolism; D010446:Peptide Fragments / Q000302:isolation & purification; D051381:Rats
D002250:Carbon Radioisotopes; D006588:Hexanones; D046911:Macromolecular Substances; D016900:Neurofilament Proteins; D009498:Neurotoxins; D010446:Peptide Fragments; C011269:2,5-hexanedione; D008239:Lysine