Isolation and characterization of the highly phosphorylated repeat domain of distinct heavy neurofilament subunit (NF-H) isoforms.
1. Recent examination of the hypothesis that distinctly phosphorylated NF-H isoforms exist in different types of neurons revealed that the extent of phosphorylation of the heavy neurofilament polypeptide of bovine ventral root motor neurons is markedly higher than that of dorsal root neurons. 2. In the present study we employed endoproteinase ASP-N for isolating the Lys-Ser-Pro (KSP)-rich domain of NF-H, which contains most of the NF-H phosphorylation sites. 3. Treatment of NF-H with ASP-N endoproteinase results in a cascade of products, the last of which is a polypeptide with apparent molecular weight of 120 kDa. Amino terminal sequence and amino acid composition analysis revealed that this fragment contains the KSP-rich domain of NF-H. 4. Treatment of ventral and dorsal root NF-H with ASP-N endoproteinase and analysis of the phosphoserine contents of the resulting 120 kDa fragments revealed that the 120 kDa fragment of ventral root NF-H is significantly more phosphorylated than that of dorsal root NF-H. 5. These findings show that the difference in extent of phosphorylation of ventral and dorsal root NF-H is due at least partly to the KSP-rich domain of NF-H.
Soussan
L
L
Department of Neurobiochemistry, George S. Wise Faculty of Life Sciences, Tel Aviv University, Israel.
Admon
A
A
Aharoni
A
A
Cohen
Y
Y
Michaelson
D M
DM
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D010450:Endopeptidases / Q000378:metabolism; D008666:Metalloendopeptidases; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009046:Motor Neurons / Q000737:chemistry; D016900:Neurofilament Proteins / Q000737:chemistry* / Q000378:metabolism; D009475:Neurons, Afferent / Q000737:chemistry; D010446:Peptide Fragments / Q000737:chemistry* / Q000302:isolation & purification; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D013126:Spinal Nerve Roots / Q000737:chemistry
D016900:Neurofilament Proteins; D010446:Peptide Fragments; C051774:neurofilament protein H; D010450:Endopeptidases; D008666:Metalloendopeptidases; C058921:endoproteinase Asp-N