Synthesis of Ribosomal Structural Proteins by Postmitochondrial Supernatant from Regenerating Rat Liver1

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Abstract
1. When the postmitochondrial supernatant (PM-supernatant) from regenerating rat liver was incubated with [3H]methionine, the incorporation of [3H]methionine into the N-terminal residues of nascent peptides on ribosomes was observed. This incorporation was sensitive to a low cocentration (2×10−6 M) of pactamycin. The results suggest that PM-supernatant has low but definite activity for the initiation of nascent protein synthesis. Polysomes and cell sap from regenerating rat liver showed negligible pactamycin-sensitive incorporation of [3H]methionine into N-terminal residues of nascent peptides. 2. PM-supernatant from regenerating rat liver was incubated with [85S]methionine in the complete reaction mixture. After addition of ribosomal proteins labelled with [3H]methionine in vivo, ribosomal structural proteins were prepared from the incubation mixture by acetic acid extraction, CM-cellulose column chromatography, Sephadex G-200 gel filtration and finally by two-dimensional acrylamide gel electrophoresis. Incorporation was observed in the greater part of ribosomal proteins on the two-dimensional gel. From the 85S-to-8H ratios of ribosomal protein fractions during the purification procedures, it appeared that the incorporation of labelled methionine into the ribosomal proteins by PM-supernatant was about 3% of that into the total proteins. When [8H]leucine was used, the values were about 4% in the same cell-free system and 5 to 6% in in vivo labelling. The results indicate that ribosomal proteins are synthesized with high efficiency by PM-supernatant from regenerating rat liver.

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