Posttranslational Protein Modification by Amino Acid Addition in Regenerating Optic Nerves of Goldfish

Abstract: Previous experiments have demonstrated that 4S RNA, (tRNA), is transported axonally during the reconnection and maturation of regenerating optic nerves of goldfish. The present experiments were performed to determine if tRNA is transported axonally during elongation of these regenerating nerves and whether, as has been demonstrated in other systems, it participates in posttranslational protein modification (PTPM). [3H]Uridine was injected into both eyes of fish with intact optic nerves and 0, 2, 4, or 8 days after bilateral optic nerve cut. Fish were killed 2 days after injection, and [3H]RNA was isolated from retinae and nerves by phenol extraction and ethanol precipitation. [3H]RNA was fractionated by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE). Although the percentage of [3H]4S RNA remained constant in all retinal and control nerve samples, regenerating nerves showed a twofold increase by 6 days after injury, suggesting that [3H]4S RNA is transported axonally in regenerating nerves as early as 6 days after injury. In other experiments, the 150,000‐g supernatant of optic nerves was analyzed for incorporation of 3H‐amino acids into proteins. No incorporation of 3H‐amino acid was found in the soluble supernatant, but when the supernatant was passed through a Sephacryl S‐200 column (removing molecules < 20,000 daltons), [3H]Arg, [3H]Lys, and [3H]Leu were incorporated into proteins. This posttranslational addition of amino acids was greater (1.4–5 times for Lys and 2–13 times for Leu) in regenerating optic nerves than nonregenerating nerves, and the growing tips of regenerating nerves incorporated 5–15 times more [3H]Lys and [3H]Leu into proteins than did the shafts. When optic axons were removed from the nerve by degeneration, most of the PTPM activity was lost, indicating a primarily intraaxonal locus for the components of the reaction. SDS‐PAGE of 3H‐modified proteins showed the presence of radioactivity in proteins of ∼13,000–18,000 38,000–46,000, and 53,000 daltons. These experiments indicate that the components necessary for PTPM by amino acids are transported axonally in regenerating optic nerves of goldfish and that the greatest activity is in the most advanced tips of the growing nerve.