Role of N-acetylamino acids in cerebral protein synthesis. I. Incorporation of radioactivity from labelled acetyl and aminoacyl moieties into protein

Abstract

Incorporation of radioactivity from [ 3H]acetate, [ 14C]aspartate, N-[ 3H-acetyl]aspartate and N-acetyl[ 14C]aspartate into cerebral proteins was investigated in vivo as well as in vitro. Concentrations of both 3H and 14C decreased rapidly in the acid soluble fraction of brain during the period following intracerebral injections of the precursors, [ 3H]acetate and [ 14C]aspartate. Concomitant with this decrease, there was an increase in the incorporation of radioactivity into protein. Only a fraction of radioactivity disappearing from the acid soluble fraction at any given interval of time was transferred to protein. However, the proportion of radioactivity incorporated into protein increased with lapse of time after injection, indicating the formation of progressively larger amounts of metabolites which could serve as more effective precursors of protein synthesis than the original injected compounds. Radioactivities from labelled acetyl and aspartyl moieties of N-acetylaspartate were also incorporated into protein. A comparison of the kinetics of incorporation of 3H and 14C from free acetate and aspartate on the one hand and from N-acetylaspartate on the other suggests that the metabolic pools that inherit acetyl and aspartyl groups from injected N-acetylaspartate are probably different from the ones in which exogenous acetate and aspartate normally enter. [ 14C]Aspartate was incorporated into protein in vitro in a reaction mixture containing cerebral polysomes pretreated with puromycin. A majority of the labelled polypeptides was released into solution under these conditions. Addition of acetate to the reaction medium appeared to stimulate the incorporation of aspartate into both soluble and particulate proteins. [ 3H]Acetate was incorporated mainly into soluble proteins, but this reaction was not dependent on the presence of polysomes and was not affected by added aspartate. The rapid uptake of [ 3H]acetate into soluble proteins and rapid loss of incorporated radioactivity on continued incubation suggest that the acetyl groups were probably added onto completed polypeptide chains in a linkage subject to high turnover. Radioactively labelled aspartate and phenylalanine were incorporated into cerebral polysomes in vitro and the extent of incorporation depended upon the concentration of Mg 2+ ions and the duration of incubation. There was no significant incorporation of N-acetylaspartate under any of the conditions tested. Acetyl-CoA slightly increased the incorporation of both aspartate and phenylalanine in the presence of low Mg 2+ ion concentration and KCl wash proteins, whereas N-acetylaspartate had no effect on this reaction.