Dephosphorylation of Neurofilaments by Exogenous Phosphatases Has No Effect on Reassembly of Subunits

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Abstract
Exhaustive in vitro dephosphorylation of porcine neurofilaments (NFs) by alkaline or acid phospha-tase did not cause a dissociation of the 210–kD (NF-H), 160–kD (NF-M), or 70–kD (NF-L) subunits and had no effect on the reassembly of NFs from urea or guanidine solution. Electron microscopy revealed that the NFs reassembled from isolated or dephosphorylated subunits had similar morphologies. Phosphatase treatment caused significant increases in the mobilities of NF-M and NF-H on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, suggesting that the subunits underwent marked conformational changes after dephosphorylation. Chemical phosphate analysis showed that as isolated NF-H, NF-M, and NF-L contained about 22, 11, and 3 mol phosphate/mol polypeptide, respectively. The corresponding values for the three subunits from alkaline phosphatase-treated NFs were about 8, 6, and 2 mol phosphate/mol polypeptide, respectively. These results indicate the occurrence of a class of phosphate moieties that is not accessible to exogenous phosphatases.