Reassembly of the 66 kD neurofilament protein in vitro following isolation and purification from bovine spinal cord.
NF-66, also known as alpha-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) M(r)NF 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 approximately 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.
Balin
B J
BJ
Department of Pathology, Medical College of Pennsylvania, Philadelphia 19129.
Miller
M E
ME
D000818:Animals; D015153:Blotting, Western; D002352:Carrier Proteins / Q000502:physiology* / Q000648:ultrastructure; D002417:Cattle; D004586:Electrophoresis; D007150:Immunohistochemistry; D066298:In Vitro Techniques; D007381:Intermediate Filament Proteins; D016900:Neurofilament Proteins / Q000096:biosynthesis*; D011506:Proteins / Q000378:metabolism*; D013116:Spinal Cord / Q000502:physiology*
D002352:Carrier Proteins; D007381:Intermediate Filament Proteins; D016900:Neurofilament Proteins; D011506:Proteins; C046643:alpha-internexin