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Reassembly of the 66 kD neurofilament protein in vitro following isolation and purification from bovine spinal cord
Author(s) -
Balin Brain J.,
Miller M. E.
Publication year - 1995
Publication title -
journal of neuroscience research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.72
H-Index - 160
eISSN - 1097-4547
pISSN - 0360-4012
DOI - 10.1002/jnr.490400109
Subject(s) - in vitro , neurofilament , spinal cord , isolation (microbiology) , chemistry , microbiology and biotechnology , biology , biochemistry , immunology , neuroscience , immunohistochemistry
NF‐66, also known as α‐internexin, has been characterized as a 66 kD mammalian neurofilament (NF) protein whose expression in developing rat brain precedes that of the low molecular weight NF protein (NF‐L). NF‐66 is thought to assemble into 10 nm diameter intermediate filaments in vitro, although the precise nature of the assembly process remains obscure. Likewise, the ability of NF‐66 to polymerize with the low (NF‐L), middle (NF‐M), and high (NF‐H) MrNF proteins has not been defined. This investigation describes the reassembly of bovine NF‐66 regarding its formation into 10 nm diameter filaments as well as its potential for polymerization with other type IV intermediate filaments. NF‐66 and the NF triplet proteins were isolated from bovine spinal cord using established biochemical extraction and isolation procedures (Balin et al., Brain Res 556:181–195, 1991), and purified by a combination of high performance liquid chromatography (HPLC) (DEAE anion exchange and hydroxylapatite column chromatography) and gel elution strategies. In vitro reassembly experiments revealed that NF‐66 formed ˜ 10 nm diameter filaments of varying length; immunoelectron microscopy demonstrated labeling of these filaments by a monoclonal antibody to intermediate filament antigen (IFA), a polyclonal antibody against rat NF‐66 and by a monoclonal antibody generated against the core region of NF‐M but cross‐reactive with NF‐66. This report is the first investigation to look at the in vitro interaction between NF‐66 and other type IV intermediate filament proteins (NF‐H, ‐M, and ‐L) and establishes that NF‐66 forms heteropolymeric filaments with these other neurofilament proteins, as confirmed by double immunolabeling. These studies suggest that NF‐66 could provide a nucleation site for the polymerization of later‐expressed proteins during neuronal development. © 1995 Wiley‐Liss, Inc.