Posttranslational modification of glycine‐extended substance P by an α‐amidating enzyme in cultured sensory neurons of dorsal root ganglia

The terminal step in the biosynthesis of substance P is the conversion of its glycine‐extended precursor to the mature, amidated peptide by the α‐amidating enzyme. This posttranslational modification was demonstrated in cultured dissociated sensory neurons of dorsal root ganglia from neonatal rats. An assay was developed to quantitate both substance P and its precursor peptide in these cells. More than 90% of these two peptide was present as mature peptide in uncultured cells. In contrast, after 8 days in culture, about 85% of the peptides was the precursor, which increased 200‐fold, whereas the level of substance P itself tripled during this culturing period. Since α‐amidating enzyme requires ascorbate for activity, this reducing agent was added to the culture medium. Ascorbate induced a dose‐dependent rise in the percentage of amidated peptide, with an apparent K m of 20μM. After 5 days of culturing in the presence of 500 μ ascorbate, substance P increased 8‐fold, constituting 70% of the total. The α‐amidating enzyme also needs copper for activity. Even with 500 μM ascorbate in the culture medium, the copper chelator diethyldithiocarbamate dose‐dependently reduced substance P synthesis by the sensory neurons, with a concomitant increase in its precursor peptide. These results suggest the presence of α‐amidating enzyme in sensory neurons of dorsal root ganglia. It is likely that conversion of other glycine‐extended precursors to their mature peptides in cell cultures would also require ascorbate and copper. © 1994 Wiley‐Liss, Inc.