Limited and selective adduction of carboxyl-terminal lysines in the high molecular weight neurofilament proteins by 2,5-hexanedione in vitro

Anthony P. DeCaprio, Jay Fowke

Research output: Contribution to journalArticle

24 Citations (Scopus)

Abstract

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° 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.

Original languageEnglish (US)
Pages (from-to)219-228
Number of pages10
JournalBrain Research
Volume586
Issue number2
DOIs
StatePublished - Jul 24 1992
Externally publishedYes

Fingerprint

Intermediate Filaments
Lysine
Neurofilament Proteins
Pyrroles
Radioactivity
Tail
Photofluorography
Proteins
Scintillation Counting
Peptides
Argon
Poisons
2,5-hexanedione
In Vitro Techniques
neurofilament protein H
Amines
Polyacrylamide Gel Electrophoresis
Spinal Cord

All Science Journal Classification (ASJC) codes

  • Neuroscience(all)
  • Molecular Biology
  • Clinical Neurology
  • Developmental Biology

Cite this

Limited and selective adduction of carboxyl-terminal lysines in the high molecular weight neurofilament proteins by 2,5-hexanedione in vitro. / DeCaprio, Anthony P.; Fowke, Jay.

In: Brain Research, Vol. 586, No. 2, 24.07.1992, p. 219-228.

Research output: Contribution to journalArticle

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