Isolation of a stable tetramer of the low molecular weight component of neurofilaments (NF-L).
When crude neurofilaments were dissolved in a solution containing 8 M urea and 1% beta-mercaptoethanol (beta-ME), the component proteins of the neurofilaments and other contaminating filaments were solubilized into monomeric forms. However, when reassembled filaments were solubilized again by the addition of urea to 8 M without beta-ME, several bands which seemed to be oligomeric forms of filament proteins were observed on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Among them, a band which appeared between microtubule-associated protein-1 (MAP-1) and fodrin was most remarkable. This band was also observed when a triplet mixture of the neurofilaments (NF-H, NF-M, NF-L) was reassembled. The molecular weight of this band was estimated to be 280 kDa. In addition, much of this component was easily isolated on DE-52 column chromatography of the reassembled crude neurofilament proteins with buffers containing 6 M urea, while the low molecular weight component of the neurofilaments (NF-L, 70 kDa) was hardly detected. Furthermore, the isolated 280 kDa component was reduced to NF-L on the addition of beta-ME to 1%. In contrast, the 280 kDa component was produced on dialysis of isolated NF-L against the assembly buffer. From these results, it is deduced that this component is the stable tetramer of NF-L which is produced through spontaneous interchain disulfide formation among protofilament tetramers.
Tokutake
S
S
Department of Molecular Biology, Psychiatric Research Institute of Tokyo.
Tanaka
K
K
D000818:Animals; D002417:Cattle; D004591:Electrophoresis, Polyacrylamide Gel; D007381:Intermediate Filament Proteins / Q000302:isolation & purification*; D007382:Intermediate Filaments / Q000378:metabolism; D008869:Microtubule-Associated Proteins / Q000302:isolation & purification; D008970:Molecular Weight; D016900:Neurofilament Proteins
D007381:Intermediate Filament Proteins; D008869:Microtubule-Associated Proteins; D016900:Neurofilament Proteins