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 e xtent of phosphorylation of the heavy neurofilament polypeptide of bov ine 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 contai ns most of the NF-H phosphorylation sites. 3. Treatment of NF-H with A SP-N endoproteinase results in a cascade of products, the last of whic h is a polypeptide with apparent molecular weight of 120 kDa. Amino te rminal sequence and amino acid composition analysis revealed that this fragment contains the KSP-rich domain of NF-H. 4. Treatment of ventra l and dorsal root NF-H with ASP-N endoproteinase and analysis of the p hosphoserine contents of the resulting 120 kDa fragments revealed that the 120 kDa fragment of ventral root NF-H is significantly more phosp horylated than that of dorsal root NF-H. 5. These findings show that t he 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.