Nerve-dependent regulation of absolute rates of protein synthesis in newt limb regenerates: Measurement of methionine specific activity in peptidyl-tRNA of the growing polypeptide chain

Abstract

Limb regeneration in the newt requires the presence of nerve fibers at the amputation surface. Regeneration does not occur upon denervation. Incorporation of [ 35S]methionine into hot TCA-precipitable material (cpm/μg of protein) increased sharply in the first few hours after denervation, then declined rapidly and plateaued at about 60% of the innervated control side at 48 hr. The specific activity of methionine was determined from hydrolyzed peptides of peptidyl-tRNA of the growing peptide chain. The hydrolyzed peptides were reacted with 3H-labeled 1-fluoro-2,4-dinitrobenzene (FDNB), and the resulting DNP-methionine was isolated by two-dimensional thin-layer chromatography. Since the specific activity of [ 3H]DNP is given, the ratio of 35S to 3H in DNP-methionine is a measure of methionine specific activity. Our results show that nervous control does not affect the specific activity of methionine in the growing polypeptide chain; the absolute rate of protein synthesis is affected. In both denervated and innervated regenerates, methionine specific activity in nascent peptides of membrane-bound polysomes was approximately half that of free polysomes, suggesting the existence of two distinct intracellular pools which are utilized for protein synthesis. Moreover, our results indicate that the specific activity of methionine in the acid soluble pool is 2–5 times lower than that found in the nascent peptides. Therefore, the acid-soluble pool cannot serve as a precursor pool for protein synthesis. We also estimated the half translation time of the nascent peptide chains. At any given moment, the nascent chain on the polysomes could be assumed to be half synthesized. Half translation time was measured as the time needed to release the labeled nascent peptides into the supernatant fluid. Denervation did not significantly affect the half translation time of average mRNA.