Preparation and some properties of rat skeletal-muscle polyribosomes.
ABSTRACT: 1. A method is described for the sucrose-gradient sedimentation analysis of ribosomes in a post-mitochondrial supernatant of rat skeletal muscle. 2. An essential feature of the method involves the use of buffer of ionic strength 0.3 for homogenization of the muscle tissue. 3. Polyribosomes can be prepared by precipitation from post-mitochondrial supernatant of skeletal muscle by adjustment of the potassium chloride content of the medium. These polyribosomes stimulate cell-free amino acid incorporation in vitro in an energy-dependent system. 4. Ribosome aggregates of uniform size distribution can be obtained by adjustment of the ionic strength of the post-mitochondrial supernatant, followed by differential sucrose-gradient centrifugation. 5. In vivo, rat skeletal-muscle polyribosomes became labelled by (14)C-labelled amino acid within 15min., and radioactivity was associated with the light ribosome species within 45min. 6. Electron microscopy of the polyribosomes revealed aggregations containing more than 40 single ribosomes.
Project description:1. The influence of hydrocortisone, insulin and diet on the size distribution of ribosomes in a post-mitochondrial supernatant prepared from rat skeletal muscle was studied by sedimentation analysis with a linear 15-40% (w/v) sucrose gradient. 2. Within 4hr. after the injection of 5mg. of hydrocortisone to well-nourished rats, a decrease in the yield per g. of muscle and proportion of total RNA due to polyribosomes was observed. Similar results were obtained in rats given a protein-free diet for 3 days before administration of the hormone. 3. Insulin injection increased the yield and proportion of polyribosomes within 2hr. and decreased the proportion of the lighter ribosomal aggregates. Similar results were noted in rats given a protein-free diet for 3 days before injection. A protein-free diet given for 3 days decreased the yield and proportion of polyribosomes. Insulin did not increase the yield of polyribosomes if rats were starved for 52hr. before injection, but decreased the yield and proportion of the lighter ribosome species. 4. A 52hr. period of starvation or 2,4-dinitrophenol (15mg./kg. body wt.) given 1hr. before the rats were killed resulted in a decreased yield and proportion of polyribosomes, and, within 6hr. of re-feeding the rats with protein-free diets, an increased concentration of polyribosomes was noted. 5. The effects of a protein-free diet, hydrocortisone and insulin on the sedimentation of muscle ribosomes were found to be in accord with their net effects on muscle protein synthesis.
Project description:1. Various subcellular fractions containing ribosomes were isolated from rat liver. 2. In the presence of [(14)C]leucine and Sephadex-treated cell sap the radioactivity incorporated into the synthesized protein resulting from the incubation of microsomal preparations or deoxycholate-treated polyribosomes was dependent on the amount of rRNA incubated. In contrast, when Sephadex-treated post-mitochondrial supernatant was incubated, the radioactivity incorporated into the synthesized protein was independent of the amount of rRNA incubated. 3. Microsomal preparations and membrane-bound ribosomes, prepared by the standard procedure, incorporated less [(14)C]leucine into protein, per mg of rRNA incubated, than free or deoxycholate-treated polyribosomes; accordingly, polyribosomes associated with the former fractions were found mainly as monomers. 4. If microsomal fractions or membrane-bound ribosomes were prepared by a simple modification of the standard procedure, i.e. by centrifugation on to a ;cushion' of 2m-sucrose, their protein-synthesizing activity was of the same order as that of the original post-mitochondrial supernatant, and membrane-free and deoxycholate-treated polyribosomes; in this case polyribosome profiles showed that very little degradation had occurred and compared well with those obtained for post-mitochondrial supernatant and isolated polyribosomes. 5. A method is described (Appendix) that provides a rapid and reliable assessment of the concentration of rRNA in subcellular fractions.
Project description:1. Polyribosomes and RNA were isolated from cultures in which tryptophanase (EC 4.2.1.-) was induced. The polyribosomes were incubated under conditions of protein synthesis, in the presence of a radioactive amino acid and a post-ribosomal supernatant fraction obtained from repressed cells. The RNA preparations were incubated under conditions of protein synthesis in the presence of a radioactive amino acid and a supernatant fraction containing ribosomes from repressed cells. 2. The system was characterized and the synthesis of a radioactive protein with the same chromatographic properties as tryptophanase was demonstrated. This synthesis was shown to be time-dependent and required the presence of RNA from induced cultures, ribosomes and an energy supply; it was inhibited by chloramphenicol. 3. The maximum activity for the synthesis of this protein was found to be associated with 23S rRNA isolated from sucrose gradients. 4. The N-terminal amino acid of tryptophanase was labelled in the protein synthesized in this system but not in the protein synthesized by polyribosomes (without added RNA). Conversely, the C-terminal amino acid of tryptophanase was labelled in the polyribosome system but not in the RNA-containing system. 5. Tryptic digests of protein labelled in vitro were compared with those of tryptophanase. No labelled tryptic peptides were identified other than tryptophanase tryptic peptides. An analysis of the results implied that in the polyribosome system almost the complete tryptophanase subunit chain was labelled but that in the RNA-containing system these chains were incompletely synthesized. 6. Sucrose-gradient analysis of protein synthesized in the RNA-containing system suggested that it cannot be converted into structures with the same sedimentation properties as native tryptophanase. 7. The significance of these results for the assay of tryptophanase mRNA and for an understanding of the control of the translation of this mRNA in vivo is discussed.
Project description:1. To investigate the role of ribosome function in regulating protein synthesis, the activity, distribution and functional states of ribosomal particles were investigated in livers of mice fed ad libitum or starved overnight. 2. The distribution of protein-synthesizing activity between polyribosomes of different sizes was analysed after incorporation of radioactive leucine, and the quantitative distribution of ribosomes as native subunits, monomers and polyribosomes was analysed after incorporation of orotic acid. Precursors labelled with (3)H or (14)C were given separately to fed and starved mice, so that livers from the two groups of animals were processed together. 3. The former experiments showed that starvation has little effect on the distribution of protein-synthesizing activity across polyribosome sedimentation patterns, though the latter experiments showed that the proportion of ribosomes existing as monomers increased from 9.5% to 15.2%, whereas the proportion existing as polyribosomes decreased from 81.4% to 75.6%. Starvation had a negligible effect on the proportion of native subunits, which accounted for 9.1% and 9.2% of the ribosomes in fed and starved mice respectively. 4. The monomeric ribosome fraction was isolated and subjected to ionic conditions which selectively dissociate single ribosomes. Starvation increased the proportion of monomers that dissociated from 59% to 72%, so the monomers that accumulate in livers of starved animals are single ribosomes and not monoribosomes resulting from degradation of polyribosomes. 5. The fate of newly formed ribosomal particles was studied by measuring the specific radioactivity of native subunits, monomers and polyribosomes at different times after injection of radioactively labelled orotic acid. Starvation did not appear to affect equilibration between newly formed particles and polyribosomes, and the radioactivity of polyribosomes in both groups of mice reached about 90% of that in native subunits after 4h. The radioactive labelling of monomers proceeded at a slower rate, especially after starvation. At 4h, the radioactivity of monomers was 64% and 55% that of native subunits in fed and starved mice respectively.
Project description:RNA molecules from nuclear and cytoplasmic polyribosomes of adenovirus-infected HeLa cells were compared by hybridization to analyse the sequence content. Nuclear polyribosomes were released by exposure of intact detergent-washed nuclei to poly(U) and purified. Cytoplasmic polyribosomes were also purified from the same cells. To show that nuclear polyribosomes contain ribosomes linked by mRNA, polyribosomes were labelled with methionine and uridine in the presence of actinomycin D in adenovirus-infected cells. Purified nuclear polyribosomes were treated with EDTA under conditions which dissociate polyribosomes into ribosomes and subunits with a simultaneous release of mRNA, and sedimented. The treatment dissociated these polyribosomes, releasing the mRNA from them. Radiolabelled total RNA from each polyribosome population was fractionated in sucrose gradients into several pools or hybridized to intact adenovirus DNA to select virus-specific RNA. Sucrose-gradient-fractionated pool-3 RNA (about 28S) and virus-specific RNA were then hybridized to fragments of adenovirus DNA cleaved by restriction endonucleases SmaI, HindIII and EcoRI by the Southern-blot technique and by filter hybridization. The results showed that nuclear RNA contained sequences, from about 0 to 18 map units, which were essentially absent from cytoplasmic RNA. Furthermore, the amount of virus-specific RNA for a particular sequence was also different in the two populations.
Project description:1. A procedure is described for the isolation of intact polyribosomes from the cytoplasm, chloroplasts and mitochondria of Euglena gracilis. 2. All three polyribosomal preparations incorporated labelled amino acids in a system in vitro. The cytoplasmic system was inhibited by chcloheximide but not by chloramphenicol. Both the chloroplast and the mitochondrial systems, however, were inhibited by chloramphenicol but not by cycloheximide. It is shown that mitochondrial polyribosomes, like the polyribosomes from cytoplasm and chloroplasts, can participate directly in protein synthesis without supplementary mRNA being added to the synthesizing system, as in previously reported instances. 3. Sedimentation coefficients were measured for the ribosomes, ribosomal subunits, and rRNA of the cytoplasm, chloroplasts and mitochondria. 4. The G+C content was 55% for cytoplasmic rRNA, 50% for chloroplast rRNA, and 29% for mitochondrial rRNA. 5. The cytoplasmic ribosomal subunits contained a ribonuclease activity that was inhibited by heparin.
Project description:1. Polyribosomes were isolated from Escherichia coli grown in media in which tryptophanase is induced and in which it is repressed. The polyribosomes from the induced bacteria had a small amount of tryptophanase activity associated with them. 2. A portion of the enzyme activity remained bound to polyribosomes during centrifuging in sucrose gradients. 3. Incubation of tryptophanase-containing polyribosomes with puromycin released enzyme activity. 4. The binding of the enzyme to the polyribosomes did not depend on the presence of DNA. 5. When the polyribosomes were incubated under conditions of protein synthesis with supernatant fraction obtained from repressed bacteria, a small but statistically significant increase in enzyme activity was produced. 6. When a radioactive amino acid was included in the incubation mixture for the tryptophanase system a radioactive protein was obtained whose chromatographic, electrophoretic and sedimentation properties were identical with those of tryptophanase. 7. The amount of incorporation was consistent with the amount of new enzyme synthesis predicted by the increase in enzyme activity. Both radioactive incorporation and increase in enzyme activity were shown to be energy-dependent and also negative controls were obtained by using zero-time incubations or polyribosomes isolated from either repressed cells or a mutant lacking the ability to produce tryptophanase. 8. The distribution of radioactive leucine in the carboxyl region of the newly labelled tryptophanase was examined by digesting the labelled protein with carboxypeptidases. It was shown that the radioactivity was more highly concentrated towards the carboxyl terminus when the incubation times for protein synthesis were shorter (implying that, with longer incubation times, longer lengths of polypeptide chain contained radioactive amino acid residues).
Project description:Exposed thiol groups do not appear to be related to the binding of (32)P-labelled polyribosomes to stripped rough endoplasmic reticulum in vitro. Treating stripped rough endoplasmic reticulum with GSSG did not diminish binding of polyribosomes, suggesting that binding in vitro has no correlation with the inhibition of protein synthesis in vitro reported by Kosower et al. (1971). Thiol reagents, which are known to dissociate ribosomes, did not significantly decrease binding of (32)P-labelled polyribosomes to stripped rough endoplasmic reticulum. Denaturing the protein of (32)P-labelled polyribosomes or stripped rough endoplasmic reticulum of liver or hepatoma with heat, trichloroacetic acid, or HClO(4) did not alter the binding in vitro. Therefore, the practice of measuring the binding of (32)P-labelled polyribosomes to stripped rough endoplasmic reticulum in vitro (Shires et al., 1971b) is an unsuitable indicator of biological significance in the intact cell.
Project description:1. Methods for the separation of membrane-bound and free ribosomes from rat brain (cortex) and skeletal muscle were described and the preparations characterized by chemical analysis and electron microscopy. The attachment of ribosomes to membranes is not an artifact of the separation procedure. 2. The rate of incorporation of l-[(14)C]leucine into protein in vitro by the membrane-bound and free ribosomes from these two predominantly non-protein-secreting tissues is compared with that by similar preparations from rat liver. With all three tissues the initial rate was higher for the membrane-bound preparations. 3. By using the technique of discharging nascent polypeptide chains by incubation with puromycin followed by treatment with sodium deoxycholate (Redman & Sabatini, 1966), a major difference was observed for the vectorial discharge of nascent protein synthesized both in vivo and in vitro on membrane-bound ribosomes from liver, on the one hand, and brain and muscle, on the other. Whereas a large part of nascent protein synthesized on membrane-bound liver ribosomes was discharged into the membranous vesicles (presumably destined for export from the cell), almost all nascent protein from membrane-bound ribosomes from brain and muscle was released directly into the supernatant. Incorporation of [(3)H]puromycin into peptidyl-[(3)H]puromycin confirmed these findings. There was thus no difference between membrane-bound and free ribosomes from brain on the one hand, and from free polyribosomes from liver on the other, as far as the vectorial release of newly synthesized protein was concerned. 4. Incubation with puromycin also showed that the nascent chains, pre-formed in vivo and in vitro, are not involved in the attachment of ribosomes to membranes of the endoplasmic reticulum. 5. The differences in vectorial discharge from membrane-bound ribosomes from liver as compared with brain and muscle are not due to the different types of messenger RNA in the different tissues. Polyphenylalanine synthesized on incubation with polyuridylic acid was handled in the same way as polypeptides synthesized with endogenous messenger. 6. It is concluded that there is a major difference in the attachment of ribosomes to the membranes of the endoplasmic reticulum of secretory and non-secretory tissues, which results in a tissue-specific difference in the vectorial discharge of nascent proteins.
Project description:1. Isolation of free and membrane-bound ribosomes from embryonic chick sternal-cartilage cells labelled for 4min with [14C]proline and their subsequent analysis for hydroxy[14C]proline indicated that cartilage procollagen biosynthesis occurs on bound ribosomes. 2. Nascent procollagen polypeptides on bound ribosomes isolated from cells labelled with [14C]lysine were found to contain hydroxy[14C]lysine indicating that hydroxylation of lysine commences while the growing chains are still attached to the ribosomes. 3. Analysis of bound ribosomes labelled with either [14C]proline or [14C]lysine on sucrose density gradients indicated that cartilage procollagen is synthesized on large polyribosomes in the range 250-400S. 4. Microsomal preparations isolated from cells pulse-labelled for 4 min with [14C]proline were used to determine the direction of release of nascent procollagen polypeptides. Puromycin induced the vectorial release of nascent procollagen polypeptides into the microsomal vesicles suggesting that the first step in the secretion of procollagen polypeptides is their transfer from the ribosomes through the membrane of the endoplasmic reticulum into the cisternal space. 5. The procollagen polypeptides secreted by cartilage cells were shown to be linked by inter-chain disulphide bonds. 6. Examination of the state of aggregation of pro-alpha chains in subcellular fractions isolated from cartilage cells labelled with [14C]proline for various periods of time have provided data on the timing and location of inter-chain disulphide-bond formation. This process commences in the rough endoplasmic reticulum after the release of completed pro-alpha chains from membrane-bound ribosomes. Pro-alpha chains isolated from fractions of smooth endoplasmic reticulum were virtually all present as disulphide-bonded aggregates, suggesting that either disulphide bonding is completed in this cellular compartment, or that procollagen needs to be in a disulphide-bonded form to be transferred to this region of the endoplasmic reticulum. 7. Comparison of these results with previously published data on disulphide bonding in tendon cells suggest that the rate of inter-chain disulphide-bond formation is significantly slower in cartilage cells.