Posttranslational modification of glycine-extended substance P by an alpha-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 alpha-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 peptides 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 alpha-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 Km of 20 microM. After 5 days of culturing in the presence of 500 microM ascorbate, substance P increased 8-fold, constituting 70% of the total. The alpha-amidating enzyme also needs copper for activity. Even with 500 microM 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 alpha-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.
Wong
M
M
Research Department Ciba-Geigy Corp., Summit, New Jersey 07901.
Jeng
A Y
AY
D000818:Animals; D000831:Animals, Newborn; D001205:Ascorbic Acid / Q000502:physiology; D002478:Cells, Cultured; D002614:Chelating Agents / Q000494:pharmacology; D003300:Copper / Q000502:physiology; D004050:Ditiocarb / Q000494:pharmacology; D005727:Ganglia, Spinal / Q000166:cytology*; D006899:Mixed Function Oxygenases / Q000378:metabolism*; D009097:Multienzyme Complexes; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D009475:Neurons, Afferent / Q000201:enzymology*; D011498:Protein Precursors / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D051381:Rats; D017207:Rats, Sprague-Dawley; D013373:Substance P / Q000031:analogs & derivatives* / Q000378:metabolism
D002614:Chelating Agents; D009097:Multienzyme Complexes; D009419:Nerve Tissue Proteins; D011498:Protein Precursors; D013373:Substance P; D003300:Copper; C046072:substance P, Gly(12)-; D004050:Ditiocarb; D006899:Mixed Function Oxygenases; C035189:peptidylglycine monooxygenase; D001205:Ascorbic Acid