Free and Membrane-bound Ribosomes of Rat Cerebral Cortex: PROTEIN SYNTHESIS IN VIVO AND IN VITRO

Abstract

Abstract Free ribosomes and membrane-bound ribosomes were prepared from cerebral cortex of 2-week-old rats using a discontinuous sucrose density gradient centrifugation technique. Analysis of protein, RNA, and phospholipid contents of these ribosomes, as well as experiments in which radioactively labeled and unlabeled ribosomes were mixed together and separated, showed that there was no more than 5% cross-contamination between free and membrane-bound ribosomes. The requirements for in vitro incorporation of amino acids were similar for the two ribosomal preparations, but the membrane-bound ribosomes showed higher incorporation activity during brief incubation periods. Our results show that a higher proportion of polysomes or heavy ribosomal aggregates in the membrane-bound ribosomes could be one of the reasons for this activity. During prolonged incubation, the capacity of membrane-bound ribosomes to incorporate amino acids declined. This was accompanied by an accumulation of undetermined heat-labile and heat-stable inhibitors into the reaction medium. A larger proportion of polypeptides was completed and released by membrane-bound ribosomes as compared to free ribosomes. The pattern of labeling of ribosomal aggregates in vivo after intracerebral injection of [3H]leucine showed that, during the period immediately following the injection, polypeptides were synthesized mainly in the heavy aggregates of both free ribosomes and membrane-bound ribosomes. Subsequently, the radioactivity was progressively transferred from the heavy aggregates to the light aggregates, and this change occurred more rapidly in the free ribosomes than in the membrane-bound ribosomes. It is suggested that, in the course of polysome turnover in vivo in the cerebral cortex, the larger aggregates could be degraded to smaller aggregates and that membrane-bound ribosomes could be less susceptible than free ribosomes to this degradation.