Post-translational modification of proteins by arginine and lysine following crush injury and during regeneration of rat sciatic nerves

Abstract

Following crush injury to rat sciatic nerves, a crude fraction of the 150,000 g supernatant can post-translationally incorporate [³H]Arg and [³H]Lys into endogenous proteins in amounts approximately 10 times uninjured control nerves. These increases occur in the proximal nerve stump within 2 h of injury and 2 weeks later in a distal segment of nerve containing the tips of the regenerating axons. In the present experiments, the endogenous nerve proteins modified by Arg or Lys in these nerve segments have been identified using two-dimensional polyacrylamide gel electrophoresis. The fraction used to assay for protein modification, the void volume of a Sephacryl S-300 column, was found to contain only a few proteins visible by Coomassie blue staining, one of which is likely to be albumin (68 kDa, pI 6.4). While this protein was modified by both Arg and Lys, the majority of label was found in areas not showing Coomassie blue staining. This indicates that of the many potential targets of post-translational arginylation and lysylation, most are proteins of relatively low abundance. A variety of proteins were modified by Arg or Lys alone while others were modified by both Arg and Lys. A high molecular weight protein (175 kDa, pI 9.0) was modified only by Lys and only at 2 h post crush. Of a variety of modified proteins of approximately 17 kDa one (pI 6.3) was modified by both Arg and Lys and at both time points, while another (pI 9.0) was modified at both time points, but only by Lys. The results show that Arg and Lys can be added post-translationally to a large number of low abundance, soluble sciatic nerve proteins, and that some of those proteins are modified only by Arg or Lys while others are modified by both Arg and Lys. Also, the modification of certain proteins appears to be associated specifically with the immediate response of a nerve to injury (e.g. 88 kDa, pI 7.1) while others are associated with the regenerative period (e.g. 56 kDa, pI 7.4).