Soluble, phosphorylated forms of the high molecular weight neurofilament protein in perikarya of cultured neuronal cells.
The high molecular weight subunit of neurofilaments (NF-H) in mouse NB2a/d1 neuroblastoma cells is extensively phosphorylated and exhibits an apparent molecular weight of 200 kDa by SDS gel electrophoresis. In this study, we observed that extensively phosphorylated NF-H variants exist as both Triton-soluble and -insoluble forms, which display different cellular distributions. Perikarya and neurites of differentiated NB2a/d1 cells were immunostained by a polyclonal antiserum (anti-NF-H) that specifically recognizes the extensively phosphorylated NF-H forms and a monoclonal antibody (SMI-31) that recognizes phosphorylated epitopes of neurofilament proteins (NFPs). When cells were extracted with Triton X-100 to remove soluble proteins, however, only axonal neurites remained immunoreactive. Immunoblot analyses established the specificity of anti-NF-H and SMI-31 and demonstrated that both Triton-soluble and -insoluble NF-H subunits exhibit an apparent molecular weight of 200 kDa. Incorporation of radiolabeled phosphate into Triton-soluble NF-H following incubation of intact NB2a/d1 cells with 32P-orthophosphate confirmed that the Triton-soluble form of NF-H is a phosphoprotein. Most NF-H subunits in the Triton-soluble fraction sedimented after centrifugation at 100,000 g for 1 h, indicating that they may be present as oligomers. The implications of these data for the development of neurofibrillary pathology are discussed.
Shea
T B
TB
Ralph Lowell Laboratories, Mailman Research Center, McLean Hospital, Belmont, MA 02178.
Majocha
R E
RE
Marotta
C A
CA
Nixon
R A
RA
D000818:Animals; D002460:Cell Line; D000939:Epitopes; D007158:Immunologic Techniques; D007381:Intermediate Filament Proteins / Q000378:metabolism*; D051379:Mice; D008970:Molecular Weight; D009447:Neuroblastoma; D016900:Neurofilament Proteins; D009474:Neurons / Q000378:metabolism*; D017830:Octoxynol; D010766:Phosphorylation; D011092:Polyethylene Glycols; D014407:Tumor Cells, Cultured / Q000378:metabolism*
D000939:Epitopes; D007381:Intermediate Filament Proteins; D016900:Neurofilament Proteins; D011092:Polyethylene Glycols; D017830:Octoxynol