Protein synthesis in BHK-21 cells infected with semliki forest virus.

[3H]leucine-labeled proteins synthesized in BHK-21 cells infected with Semliki Forest virus were fractionated by polyacrylamide gel electrophoresis (PAGE). Cellular and virus-specific proteins were identified by difference analysis of the PAGE profiles. The specific activity of intracellular [3H-A1leucine was determined. Two alterations of protein synthesis, which develop with different time courses, were discerned. (i) In infected cultures an inhibition of overall protein synthesis to about 25% of the protein synthesis in mock-infected cultures develops between about 1 and 4 h postinfection (p.i.). (ii) The relative amount of virus-specific polypeptides versus cellular polypeptides increases after infection. About 80% of the proteins synthesized at 4 h p.i. are cellular proteins. Since significant amounts of nontranslocating robosomes in polyribosomes were not detected up to 7 h p.i., the inhibition of protein synthesis is not caused by inactivation of about 75% of all polyribosomes but by a decreased protein synthetic activity of the majority of polyribosomes. Indirect evidence indicates that an inhibition of elongation and/or release of protein synthesis develops in infected cells, which is sufficient to account for the observed inhibition of protein synthesis. Inhibition of over-all protein synthesis developed when virus-specific RNA began to accumulate at the maximal rate. This relationship was observed during virus multiplication at 37, 30, and 25 C. A possible mechanism by which synthesis of virus-specific RNA in the cytoplasm could inhibit cellular protein synthesis is discussed. Indirect evidence and analysis of polyribosomal RNA show that the increased synthesis of virus-specific protein is brought about by a substitution of cellular by viral mRNA in the polyribosomes.     

Protein Synthesis in BHK-21 Cells Infected with Semliki Forest Virus

[³H]leucine-labeled proteins synthesized in BHK-21 cells infected with Semliki Forest virus were fractionated by polyacrylamide gel electrophoresis (PAGE). Cellular and virus-specific proteins were identified by difference analysis of the PAGE profiles. The specific activity of intracellular [³H]leucine was determined. Two alterations of protein synthesis, which develop with different time courses, were discerned. (i) In infected cultures an inhibition of overall protein synthesis to about 25% of the protein synthesis in mock-infected cultures develops between about 1 and 4 h postinfection (p.i.). (ii) The relative amount of virus-specific polypeptides versus cellular polypeptides increases after infection. About 80% of the proteins synthesized at 4 h p.i. are cellular proteins. Since significant amounts of nontranslocating ribosomes in polyribosomes were not detected up to 7 h p.i., the inhibition of protein synthesis is not caused by inactivation of about 75% of all polyribosomes but by a decreased protein synthetic activity of the majority of polyribosomes. Indirect evidence indicates that an inhibition of elongation and/or release of protein synthesis develops in infected cells, which is sufficient to account for the observed inhibition of protein synthesis. Inhibition of over-all protein synthesis developed when virus-specific RNA began to accumulate at the maximal rate. This relationship was observed during virus multiplication at 37, 30, and 25 C. A possible mechanism by which synthesis of virus-specific RNA in the cytoplasm could inhibit cellular protein synthesis is discussed. Indirect evidence and analysis of polyribosomal RNA show that the increased synthesis of virus-specific protein is brought about by a substitution of cellular by viral mRNA in the polyribosomes.     

The effect of phenobarbitone on protein synthesis by liver polyribosomes in fed and starved rats.

1. The effect of phenobarbitone on the rate of protein synthesis and on the sedimentation patterns of various liver subcellular fractions containing ribosomes was studied in rats. 2. Phenobarbitone treatment increased the incorporation of [114C]leucine into protein by all preparations, provided they had not been subjected to preliminary treatment with Sephadex G-25. The phenobarbitone-induced effect on incorporation was associated with a gain in liver weight and a higher degree of polyribosomal aggregation. 3. Preparations that were treated with Sephadex G-25 incorporated more radioactivity into protein, but did not show the response to phenobarbitone treatment. 4. When the influence of starvation and phenobarbitone was studied separately on membrane-bound and membrane-free polyribosomes, it was shown that whereas both classes of polyribosomes were affected by starvation, apparently only the former class was susceptible to phenobarbitone stimulation of protein synthesis. 5. The decreased capacity for protein synthesis of polyribosomes from starved rats was independent of their association with the membranes of the endoplasmic reticulum, but resulted from polyribosomal disaggregation, from an intrinsic defect of the polyribosomes themselves and from changes in composition of the cell cap. 6. The results are discussed in relation to the problem of the control of protein biosynthesis and of the functional separation of membrane-bound and membrane-free polyribosomes.     

Effect of exercise on synthesis and degradation of muscle protein.

Several reports have shown that amino acid utilization via oxidation and gluconeogenesis is increased during exercise. The purpose of this study was to investigate whether these changes are accompanied by alterations in protein synthesis and degradation in the muscle of exercising rats. One group of rats was made in swim for 1h and then protein synthesis and protein degradation were measured in a perfused hemicorpus preparation. Protein synthesis was decreased and protein degradation was increased in exercised rats compared with sedentary control rats. Exercise also decreased amino acid incorporation by isolated polyribosomes from muscle. Measurement of several muscle proteinase activities demonstrated that exercise had no effect on alkaline proteinase or Ca2+-activated proteinase. However, the free (unbound) cathepsin D activity was elevated in muscle of exercised rats, whereas the total activity of catepsin D was unchanged. This increase in the proportion of free cathepsin D activity suggests that lysosomal enzymes may be involved in the increased protein degradation that was observed.     

Isolation of preparative amounts of polyribosomes from normal rabbit and guinea pig spleen]

Preparative amounts of polyribosomes were isolated from normal rabbit and guinea pig spleen; up to 40 optical units of the polyribosome preparation could be obtained by centrifugation in a Spinco L-2B centrifuge with SW-27 rotor. The amount of polyribosomes isolated from spleens of immune animals was 2-3 higher than that isolated from normal animal spleens. Concentration of polyribosomal preparations by lyophylization and the storage of dried preparations do not alter the sedimentation properties of the polyribosomes. The distribution pattern of normal rabbit spleen polyribosomes in a linear sucrose gradient and the sedimentation constants of the polyribosome peaks are in good agreement with data reported by some other authors for plasmocytome polyribosomes. Using electrophoresis in agarose-polyacrylamide gel the radioactive proteins synthesized in the cell culture of normal rabbit spleen it was shown that in normal spleen the average amount of globulins makes up to 35% of total protein synthesis, as reported by some authors.     

Effect of tryptophan on livers of pregnant and lactating rats and their fetuses and pups

The effect of the administration of L-tryptophan on hepatic polyribosomes and protein synthesis in pregnant rats and their fetuses and in lactating rats and their pups was investigated. Pregnant rats tube-fed tryptophan 1 hr before killing revealed increased hepatic protein synthesis but essentially unmodified polyribosomal aggregation of maternal livers while no changes were observed in fetal livers in comparison to controls (water-treated). Lactating rats tube-fed tryptophan 1 hr before killing revealed increased polyribosomal aggregation and protein synthesis of the livers in comparison to controls. Pups of these mothers that received tryptophan intraperitoneally 1 hr before killing did not reveal a significant change in the hepatic polyribosomes or protein synthesis.     

Inhibition of host protein synthesis in vaccinia virus-infected cells in the presence of cordycepin (3''-deoxyadenosine).

Cordycepin inhibited efficiently viral mRNA and polyadenylic acid syntheses in vaccinia virus-infected cells, but allowed the shutoff of host protein synthesis to occur. Therefore, cordycepin was used to study this shutoff in the absence of gene expression. Ribosome transit time was increased in infected cells, revealing an inhibition at the level of elongation and/or release of polypeptide chains. However, the disappearance of heavy polysomes in vaccinia virus-infected cells showed that the inhibition of host protein synthesis resulted predominantly from a block at the stage of initiation. This conclusion was confirmed by the recovery of heavy polyribosomes when low levels of cycloheximide were added to slow down ribosome release from the mRNA. Similar amounts of cellular mRNA (present in the polyribosomes) were found in vaccinia virus-infected cells and in mock-infected cels (exposed to cordycepin), showing that the cellular mRNA was not inactivated in these conditions. It was concluded that a component of the vaccinia virion inhibits, in the absence of viral RNA and polyadenylic acid syntheses, host protein synthesis at the level of initiation and, to a lesser extent, at the level of elongation (and/or release) of polypeptide chains.     

Alterations in the protein synthetic apparatus of Friend erythroleukemia cells during their erythroid differentiation

The changes in rate of protein synthesis and cell division and the distribution of polyribosomes and globin mRNA on the polyribosomes of Friend erythroleukemia (FL) cells exposed to 2% DMSO and maintained at low cell density, were examined at different times after exposure to DMSO. The rate of protein synthesis and the capacity of cells to divide declined in concert to 50% of the level found in untreated cell cultures at 24 hours after exposure. Thereafter these rates recovered to 70% of the rate found in untreated control cultures until 96 hours post-exposure and then irreversibly declined as the cells lost the capacity to divide. The proportion of ribosomes present as polyribosomes in cells exposed to DMSO paralleled the capacity of these cells to synthesize protein. The distribution of polyribosomes analyzed by sedimentation in sucrose gradients demonstrated that a discrete, abundant class of polyribosomes composed of pentamers to heptamers appeared as early as 48 hours after exposure to DMSO. The appearance of an abundant class of polyribosomes was correlated with globin synthesis by demonstrating that a discrete class of polyribosomes arises in cells treated with the inducers hexamethylene bisacetamide and hemin.     

Inhibition of protein synthesis by N-methyl-N-nitrosourea in vivo

1. The intraperitoneal injection of N-methyl-N-nitrosourea (100mg/kg) caused a partial inhibition of protein synthesis in several organs of the rat, the maximum effect occurring after 2-3h. 2. In the liver the inhibition of protein synthesis was paralleled by a marked disaggregation of polyribosomes and an increase in ribosome monomers and ribosomal subunits. No significant breakdown of polyribosomes was found in adult rat brains although N-methyl-N-nitrosourea inhibited cerebral and hepatic protein synthesis to a similar extent. In weanling rats N-methyl-N-nitrosourea caused a shift in the cerebral polyribosome profile similar to but less marked than that in rat liver. 3. Reaction of polyribosomal RNA with N-[(14)C]methyl-N-nitrosourea in vitro did not lead to a disaggregation of polyribosomes although the amounts of 7-methylguanine produced were up to twenty times higher than those found after administration of sublethal doses in vivo. 4. It was concluded that changes in the polyribosome profile induced by N-methyl-N-nitrosourea may reflect the mechanism of inhibition of protein synthesis rather than being a direct consequence of the methylation of polyribosomal mRNA.     

Initiation factors in protein synthesis by free and membrane-bound polyribosomes of liver and hepatoma.

The activity of initiation factors obtained from free and membrane-bound polyribosomes of liver and of transplantable H5123 hepatoma of rats was investigated by using an assay of protein synthesis in vitro in which poly (U)-directed polyphenylalanine synthesis was measured. Initiation factors of membrane-bound polyribosomes prepared by using the anionic detergent deoxycholate exhibited less activity in incorporating [14C]phenylalanyltRNA into polypetides than did initiation factors of free polyribosomes. However, when membrane-bound polyribosomes were prepared after using the non-ionic detergent Triton X-100, no significant differences in activities in polyphenylalanine synthesis were observed between the initiation factors of free and membrane-bound polyribosomes. These results suggest that Triton X-100 is preferable to deoxycholate in the isolation of of initiation factors from polyribosomes. Initiation factors, prepared by using Triton X-100, of free polyribosomes of hepatoma exhibited greater activity in the stimulation of polyphenylalanine synthesis than did the initiation factors of free or membrane-bound polyribosomes of host livers or of membrane-bound polyribosomes of hepatomas.     

Polyribosomes redistribute from dendritic shafts into spines with enlarged synapses during LTP in developing rat hippocampal slices

The presence of polyribosomes in dendritic spines suggests a potential involvement of local protein synthesis in the modification of synapses. Dendritic spine and synapse ultrastructure were compared after low-frequency control or tetanic stimulation in hippocampal slices from postnatal day (P)15 rats. The percentage of spines containing polyribosomes increased from 12% +/- 4% after control stimulation to 39% +/- 4% after tetanic stimulation, with a commensurate loss of polyribosomes from dendritic shafts at 2 hr posttetanus. Postsynaptic densities on spines containing polyribosomes were larger after tetanic stimulation. Local protein synthesis might therefore serve to stabilize stimulation-induced growth of the postsynaptic density. Furthermore, coincident polyribosomes and synapse enlargement might indicate spines that are expressing long-term potentiation induced by tetanic stimulation.     

Fraction of Ribosomes Synthesizing Protein as a Function of Specific Growth Rate

The fraction of ribosomes engaged in protein synthesis in Escherichia coli growing at specific growth rates ranging from 0.3 to 1.2 h(-1) was estimated from measurements of nascent protein/ribosome and of polyribosomes. Both measurements showed that the fraction of ribosomes synthesizing protein increased with specific growth rate, from 30 to 70% over the range studied. Polyribosome measurements made at different specific growth rates in four E. coli strains showed no significant differences between strains. The increase in the fraction of polyribosomes had begun within 30 s after a shift-up from glucose-minimal medium, and was complete within 2 to 5 min. These results indicate that the function of ribosomes is regulated.     

Inhibitory action of erythromycin on protein biosynthesis by isolated polyribosomes

Consistent with the previous work by Pestka (Antimicrob. Agents Chemother.5, 255, 1974) on the binding of erythromycin to polyribosomes, we found that erythromycin does not inhibit protein synthesis catalyzed by polyribosomes. This is due to the presence of nascent peptidyl tRNA on the naturally occurring polyribosomes. In a soluble extract from E. coli pretreated to remove the ribosome releasing factor, polyribosomes without nascent polypeptides remain intact and can catalyze protein synthesis in the absence of initiation. In this system erythromycin effectively inhibited protein synthesis. The inhibition by erythromycin was caused by premature release of oligopeptidyl tRNA from polyribosomes.     

The rate-limiting step of protein synthesis in vivo and in vitro and the distribution of growing peptides between the puromycinlabile and puromycin-non-labile sites on polyribosomes

1. At 3 min after an intravenous injection of radioactive amino acids into the rat, the bulk of radioactivity associated with liver polyribosomes can be interpreted as growing peptides. 2. In an attempt to identify the rate-limiting step of protein synthesis in vivo and in vitro, use was made of the action of puromycin at 0 degrees C, in releasing growing peptides only from the donor site, to study the distribution of growing peptides between the donor and acceptor sites. 3. Evidence is presented that all growing peptides in a population of liver polyribosomes labelled in vivo are similarly distributed between the donor and acceptor sites, and that the proportion released by puromycin is not an artifact of methodology. 4. The proportion released by puromycin is about 50% for both liver and muscle polyribosomes labelled in vivo, suggesting that neither the availability nor binding of aminoacyl-tRNA nor peptide bond synthesis nor translocation can limit the rate of protein synthesis in vivo. Attempts to alter this by starvation, hypophysectomy, growth hormone, alloxan, insulin and partial hepatectomy were unsuccessful. 5. Growing peptides on liver polyribosomes labelled in a cell-free system in vitro or by incubating hemidiaphragms in vitro were largely in the donor site, suggesting that either the availability or binding of aminoacyl-tRNA, or peptide bond synthesis, must be rate limiting in vitro and that the rate-limiting step differs from that in vivo. 6. Neither in vivo nor in the hemidiaphragm system in vitro was a correlation found between the proportion of growing peptides in the donor site and changes in the rate of incorporation of radioactivity into protein. This could indicate that the intracellular concentration of amino acids or aminoacyl-tRNA limits the rate of protein synthesis and that the increased incorporation results from a rise to a higher but still suboptimum concentration.     

Protein biosynthesis in a homologous, cell-free system in the presence of chick embryo RNA isolated from free and membrane-bound polyribosomes.

Protein synthesis has been studied in a cell-free system from chick embryo, in the presence of homologous RNA isolated from free and endoplasmic reticulum-bound polyribosomes. The two RNA fractions showed equal activities in total protein synthesis. However, while the RNA from bound polyribosomes mainly supported synthesis of high molecular weight, TCA-insoluble polypeptides, the RNA from free polyribosomes was more active in the synthesis of low molecular weight, TCA-soluble polypeptides. Optimal conditions for translation of the two RNA's under study were different when studied in a cell-free system with reduced content of endogenous matrix. Collagen synthesized in the system was identified by collagenase digestion. Collagen synthesis was demonstrated only in the presence of RNA from endoplasmic reticulum-bound polyribosomes, and represented 16-19% of total protein synthesis.     

Trichodermin,a possible inhibitor of the termination process of protein synthesis

The mode of action of the antibiotic, trichodermin, on yeast cells has been investigated. Trichodermin specifically inhibits protein synthesis and, during the in vivo inhibition of protein synthesis, ribosomes remain in polyribosomes rather than shifting to monoribosomes. This observation suggests that trichodermin inhibits either an elongation step or a termination step of protein biosynthesis. These two possibilities were distinguished by comparing the action of trichodermin with that of cycloheximide, a known elongation inhibitor, upon the reformation of polyribosomes during recovery from a block in polypeptide chain initiation. Cycloheximide slows the recovery of polyribosomes from monoribosomes following a block in polypeptide chain initiation whereas trichodermin enhances the recovery of polyribosomes. This observation is interpreted to mean that trichodermin primarily inhibits the termination step of protein biosynthesis.     

The response of the small intestine to vitamin D. Isolation and properties of chick intestinal polyribosomes

Undegraded polyribosome preparations may be obtained from chick intestinal mucosa if ribonuclease activity is strictly controlled. This is best achieved by homogenization of the mucosa directly in rat liver cell-sap. 2. The extent of amino acid incorporation by chick intestinal polyribosomes is greatly influenced by the source of the cell-sap. Sephadex-treated intestinal cell-sap caused impaired incorporation and release of completed polypeptide chains, whereas Sephadex-treated rat liver cell-sap promoted the polymerization of up to 90 amino acids per ribosome. Under optimum conditions 30-35% of the nascent polypeptide chains are completed and released. 3. The preparation of an antiserum against the calcium-binding protein formed in response to vitamin D is described. It is shown that the antiserum is highly specific for calcium-binding protein. 4. This antiserum was used to investigate the ability of chick intestinal polyribosomes to synthesize calciumbinding protein. Only polyribosomes from chicks receiving vitamin D have the ability to synthesize calcium-binding protein. Moreover, the product formed in vitro has the same electrophoretic mobility as calcium-binding protein synthesized in vivo. 5. It is concluded that one of the main functions of vitamin D in the small intestine is to induce the synthesis de novo of calcium-binding protein.     

In vitro translation of cytosolic and peroxisomal epoxide hydrolase and catalase on liver polyribosomes from untreated and clofibrate-treated C57B1/6 mice

Free and membrane-bound polyribosomes were isolated from the livers of untreated and clofibrate-treated male C57B1/6 mice. The in vitro translation products were investigated in a rabbit reticulocyte cell-free system by immunoprecipitation of cytosolic epoxide hydrolase, catalase and albumin. The soluble forms of epoxide hydrolase present in cytosol and in peroxisomes were found to be synthesized on free polyribosomes and could not be distinguished from each other, since only one primary translation product was found with the methods used. Clofibrate treatment was found to increase total protein synthesis, synthesis of soluble epoxide hydrolase and translational efficiency of the isolated polyribosomes.