Hypophosphorylated neurofilament subunits in the cytoskeletal and soluble fractions of cultured bovine adrenal chromaffin cells.
The neurofilament proteins in cultured bovine adrenal chromaffin cells are in a hypophosphorylated state, as determined by the co-migration of the 160,000 and 210,000 molecular weight subunits with in vitro dephosphorylated bovine brain subunits on sodium dodecyl sulfate polyacrylamide gels. In addition, chromaffin cells were not stained by anti-heavy neurofilament subunit that binds only to phosphorylated epitopes. Pulse-labeling with 32Pi in the presence and absence of the protein synthesis inhibitor emetine indicated that some neurofilament protein phosphorylation occurred co-translationally and/or immediately after synthesis of the proteins. Pulse-chase experiments showed that the three neurofilament proteins rapidly attained their maximal phosphorylation levels, as multiple forms of either of the respective subunits were not seen after a one hour chase. We found that Triton X-100-soluble forms of high molecular weight neurofilament and middle molecular weight neurofilament subunits were present in chromaffin cells, and they also co-migrated with standard neurofilament proteins dephosphorylated in vitro. However, there were differences between the phosphopeptide maps of cytoskeleton-associated and soluble middle molecular weight neurofilament subunit, suggesting that the localization of phosphate moieties rather than extent of phosphorylation influences the association of the subunit with neurofilaments. Double immunofluorescence staining of cell cultures with antibody to the 70,000 molecular weight subunit and with anti-vimentin showed that chromaffin cells do not express vimentin.
D000311:Adrenal Glands / Q000166:cytology / Q000378:metabolism* / Q000648:ultrastructure; D000818:Animals; D000906:Antibodies / Q000276:immunology; D002417:Cattle; D002478:Cells, Cultured; D002838:Chromaffin System / Q000166:cytology / Q000378:metabolism* / Q000648:ultrastructure; D003599:Cytoskeleton / Q000378:metabolism*; D007381:Intermediate Filament Proteins / Q000276:immunology / Q000378:metabolism*; D016900:Neurofilament Proteins; D017830:Octoxynol; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D011092:Polyethylene Glycols; D012995:Solubility; D013347:Subcellular Fractions / Q000378:metabolism
D000906:Antibodies; D007381:Intermediate Filament Proteins; D016900:Neurofilament Proteins; D010761:Phosphorus Radioisotopes; D011092:Polyethylene Glycols; D017830:Octoxynol