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 filame nts in vitro, although the precise nature of the assembly process rema ins 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 d efined, This investigation describes the reassembly of bovine NF-66 re garding its formation into 10 nm diameter filaments as well as its pot ential for polymerization with other type PV intermediate filaments, N F-66 and the NF triplet proteins were isolated from bovine spinal cord using established biochemical extraction and isolation procedures (Ba lin et al,, Brain Res 556:181-195, 1991), and purified by a combinatio n of high performance liquid chromatography (HPLC) (DEAE anion exchang e and hydroxylapatite column chromatography) and gel elution strategie s, In vitro reassembly experiments revealed that NF-66 formed similar to 10 nm diameter filaments of varying length; immunoelectron microsco py demonstrated labeling of these filaments by a monoclonal antibody t o intermediate filament antigen (IFA), a polyclonal antibody against r at NF-66 and by a monoclonal antibody generated against the core regio n of NF-M but cross-reactive with NF-66, This report is the first inve stigation to look at the in vitro interaction between NF-66 and other type IV intermediate filament proteins (NF-H, -M, and -L) and establis hes that NF-66 forms heteropolymeric filaments with these other neurof ilament proteins, as confirmed by double immunolabeling. These studies suggest that NF-66 could provide a nucleation site for the polymeriza tion of later-expressed proteins during neuronal development. (C) 1995 Wiley-Liss, Inc.