Elsevier

Brain Research

Volume 586, Issue 2, 24 July 1992, Pages 219-228
Brain Research

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

https://doi.org/10.1016/0006-8993(92)91630-WGet rights and content

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.

References (48)

  • A.A. Rashin et al.

    On the environment of ionizable groups in globular proteins

    J. Mol. Biol.

    (1984)
  • L.M. Sayre et al.

    Pathogenesis of experimental giant neurofilamentous axonopathies: a unified hypothesis based on chemical modification of neurofilaments

    Brain Res. Rev.

    (1985)
  • L.M. Sayre et al.

    Structural basis of gamma-diketone neurotoxicity: non-neurotoxicity of 3,3-dimethyl-2,5-hexanedione, a gamma-diketone incapable of pyrrole formation

    Toxicol. Appl. Pharmacol.

    (1986)
  • G. Shaw

    Identification of previously unrecognized sequence motifs at the extreme carboxyterminus of the neurofilament subunit NF-M

    Biochem. Biophys. Res. Commun.

    (1989)
  • D.C. Anthony et al.

    The effect of 3,4-dimethyl substitution on the neurotoxicity of 2,5-hexanedione. II. Dimethyl substitution accelerates pyrrole formation and protein crosslinking

    Toxicol. Appl. Pharmacol.

    (1983)
  • R.O. Beauchamp et al.

    A critical review of the literature on carbon disulfide toxicity

    CRC Crit. Rev. Toxicol.

    (1984)
  • M.J. Carden et al.

    2,5-Hexanedione neuropathy is associated with the covalent crosslinking of neurofilament proteins

    Neurochem. Pathol.

    (1986)
  • S.S.M. Chin et al.

    ]xpression of rat neurofilament proteins NF-L and NF-M in transfected non-neuronal cells

    Eur. J. Cell Biol.

    (1989)
  • S.S.M. Chin et al.

    Transfected rat high-molecular-weight neurofilament (NF-H) coassembles with vimentin in a predominantly nonphosphorylated form

    J. Neurosci.

    (1990)
  • A.W. Clark et al.

    The axonal pathology in chronic IDPN intoxication

    J. Neuropathol. Exp. Neurol.

    (1980)
  • S.A. Cohen et al.
  • A.P. DeCaprio

    n-Hexane neurotoxicity: a mechanism involving pyrrole adduct formation in axonal cytoskeletal protein

    Neurotoxicology

    (1987)
  • A.P. DeCaprio et al.

    Mechanisms of formation and quantitation of imines, pyrroles, and stable non-pyrrole adducts in 2,5-hexanedione-treated protein

    Mol. Pharmacol.

    (1987)
  • J. Eyer et al.

    Influence of the phosphorylation state of neurofilament proteins on the interactions between purified filaments in vitro

    Biochem. J.

    (1988)
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