Acetylation of Ribosomal Proteins in Regenerating Rat Liver

It has been shown that rat liver ribosomal proteins can be acetylated both in viuo and in vitro (Liew & Gornall, 1973). After purification of the ribosomal proteins to remove any possible contamination by lipids, nucleotides and cytoplasmic protein, the radioactivity still remained in these proteins. When the [’Hlacetate-labelled ribosomal proteins were hydrolysed in strong acid at 110°C overnight, more than 50% of the radioactivity was removed. These results are regarded as evidence that the labile acetyl groups were incorporated into the ribosomal proteins. Acetylation of ribosomal proteins was found to occur also in rat heart, kidney and thymus gland and in rabbit reticulocytes. The present report deals with the possible direct relationship between acetylation and protein biosynthesis in regenerating rat liver. It is known that protein synthesis is increased after partial hepatectomy and reaches a maximum rate after approx. 15-24 h (Tsukada et al., 1968). Partial hepatectomy or sham operation was performed in groups of rats according to the following experimental design. [’HIAcetate (5 mCi) was given to rats that had been partially hepatectomized for 1, 3,6, 14 or 24h. Then, 15 min after the administration of the tracer, the animals were killed and the ribosomes were isolated as previously described (Liew & Korner, 1970). Ribosomal proteins were obtained by extraction with LiCl-urea. An increased incorporation of [’Hlacetate into ribosomal proteins occurred at 3,6, 14 and 24h as compared with the sham-operated animals, but not in the partially hepatectomized rats after 1h. A maximum twofold increase in acetylation of ribosomal proteins over the sham-operated controls was observed 6 h after partial hepatectomy. Ribosomes obtained 3 and 6 h after partial hepatectomy were separated into ‘total’, ‘free’, and ‘bound’ fractions (Redman, 1969). It was found that acetylation of the ribosomal proteins was significantly affected by partial hepatectomy in all three fractions. In double-isotope-labelling experiments [’Hlacetate and [14C]serine were given to rats that had been partially hepatectomized for 3,6, 14 and 24h and ribosomes were isolated 15min after the administration of the tracers. It was found that the incorporation of [14C]serine into the ‘total’ ribosomal proteins was increased to 167 % of sham-operated control values at 24h and that [’Hlacetate incorporation was greatest 6h after partial hepatectomy. These observations suggest that acetylation of ribosomal proteins occurs before an increase of protein synthesis. Further analysis was carried out to separate nascent chains that were attached to the ‘total’, ‘bound’ and ‘free’ ribosomes by treatment in uitro with either puromycin or EDTA. Ribosomal subunits sedimented through 1 M-sucrose during 198 OOOg centrifugation overnight. Both the nascent chains and the ribosomal subunits were then treated with NaOH before Millipore filtration to remove any possible contamination by RNA. It was found that [3H]acetate and [14C]serine were incorporated into both the nascent chains and ribosomal subunits. The labelled ribosomal proteins were then treated with trypsin and proteases. The amino acid residues in the hydrolysate were separated by exclusion chromatography and identified by ion-exchange chromatography as described previously (Liew et al., 1970). Three labelled amino acid residues were identified as Nu-acetylserine, Na-acetyl-lysine and N”-acetyl-lysine. In uiuo the administration of puromycin to rats 6 h after partially hepatectomy, before the injection of the tracers, resulted in a 90% inhibition of [14C]serine incorporation into the ribosomal proteins and a concomitant 60% inhibition of the incorporation of [3H]acetate into ribosomal proteins. These results suggest that acetylation of ribosomal proteins may be closely associated with the regulation of protein synthesis. This work was supported by Medical Research Council and Ontario Heart Foundation, Canada.