The posttranslational modification of endogenous proteins by the addition of a variety of amino acids occurs in axons of both vertebrate and invertebrate nerves. The activity of these reactions increases up to 100 fold following injury of the nerve. We are investigating these reactions with the long term goal of better understanding the biochemical events which occur in damaged axons, and identifying why certain nerves fail to regenerate following injury. The specific goals of the proposed experiments are to: 1) use biochemical procedures to determine if transfer RNA is the amino acid donor to the acceptor proteins. 2) Determine the site of addition (terminal, side chain) of the amino acid to the acceptor protein, using specific hydrolyzing enzymes, and HPLC and amino acid sequencing techniques. 3) To identify the targeted proteins by one and two dimensional gel electrophoresis in intact and regenerating rat sciatic and goldfish optic nerves. In these experiments, attempts will be made to identify modified proteins associated with slow component b in both nerves and synaptic terminals of goldfish optic nerves. 4) Examine protein modification of a wider range of proteins, in squid axoplasm. Axoplasm obtained from the giant axon of the squid can be analyzed free of contamination from supporting cells, and therefore is an excellent source for monitoring axon specific protein modification. 5) Demonstrate the reaction in vivo by use of injections of 3H amino acids directly in the rat sciatic nerve, followed by light (and possibly EM) autoradiography, and polyacrylamide gel electrophoresis. Further study of this reaction will yield important information concerning a novel biochemical reaction which occurs in nerve axons. Since this reaction is increased so dramatically following injury, it should also contribute to our understanding of the role these reactions play in nerve regeneration.
Showing the most recent 10 out of 12 publications
