Differences in size, structure and function of free and membrane-bound polyribosomes of rat liver. Evidence for a single class of membrane-bound polyribosomes

Abstract

Free loosely bound and tightly bound polyribosomes were separated from rat liver homogenate by salt extraction followed by differential centrifugation, and several of their structural and functional properties were compared to resolve the existence of loosely bound polyribosomes and verify the specificity of the separation. The free and loosely bound polyribosomes have similar sedimentation profiles and polyribosome contents, their subunit proteins have similar electrophoretic patterns and their products of protein synthesis in vitro show a close correspondence in size and amounts synthesized. In contrast, the tightly bound polyribosomes have different properties from those of the free and loosely bound polyribosomes; their average size is significantly smaller; their polyribosome content is higher; their 60S-subunit proteins lack two components and contain four or more components not found elsewhere; their products of protein synthesis in vitro differ in size and amounts synthesized. These observations show that rat liver membranes entrap a large fraction of the free polyribosomes at low salt concentrations and that these polyribosomes are similar to those ofthe free-polyribosome fraction and are different from those of the tightly bound polyribosome fraction in size, structure and function.

Distribution of Hepatic Ribosomes between Different Functional States in Well-Fed and Protein-Energy-Deficient Rats

“…Ho mogenization of the tissue in high-KCl me dium reduced the loss of RNA to 12%, and this could be attributable to release of loosely bound ribosomes/polysomes from the mem branes, as earlier reported by other investiga tors [19]. Since no less than about 90% of the free ribosomal population is usually recov ered in the PMS [4,5], our data (table 1) sug gested that protein-energy malnutrition in rats did not lead to degranulation of the RER with increase in the pool of free ribosomes.…”

“…Earlier reports [4] put the ratio of free to membrane-bound ribosomes in rat liver as 1:3, but more recent studies suggest that the ratio is 1:2 [19]. This minor discrepancy not withstanding, it is clear that membrane-at tached ribosomes constitute the main source of liver RNA.…”

Synthesis and insertion of cytochrome P-450 into endoplasmic reticulum membranes.

“…Because the ratio of bound to free polysomes is 2:1 in rat liver (38), our results suggest that, in the hepatocyte, cytochrome P-450 is synthesized exclusively (>95%) by polysomes associated with ER membranes. A similar conclusion has previously been reached from studies of the distribution of in vivo synthesized nascent cytochrome P-450 peptide chains in free and bound polysomes (39,40).…”

Molecular cloning and nucleotide sequence of cDNA for rat ornithine carbamoyltransferase precursor.

“…Details of preparation of rat liver free polysomes and polysome immunoprecipitation will be described elsewhere. Briefly, free polysomes were first separated from membrane-bound polysomes by differential centrifugation (18) and then collected by magnesium precipitation (19). Polysomes synthesizing ornithine carbamoyltransferase were precipitated with anti-omnithine carbamoyltransferase IgG and formalin-fixed Staphylococcus aureus cells (20).…”