The isolation and properties of cardiac ribosomes and polysomes

1. A method is described by which good yields of ribosomes and polysomes free of contamination by submitochondrial fragments can be prepared from rat cardiac muscle. These preparations are capable of incorporation of amino acids into protein in vitro. 2. The ribosome preparation consists of 32% of monomeric ribosomes and 68% of ribosomal aggregates or polysomes. The polysome preparation has a decreased monomeric content. Dimers, trimers, tetramers, pentamers and larger components can be differentiated. 3. The polysome aggregate structure is degraded to monomeric ribosomes on incubation with small amounts of ribonuclease or by preparation in the absence of Mg²⁺ ions. The degradation in the absence of Mg²⁺ ions was not reversible and drastically decreased the incorporation of amino acids in vitro. 4. The cardiac ribosomes contained two major RNA species sedimenting at 19s and 28s in a 1:2·4 ratio. 5. The RNA/protein ratio of cardiac ribosomes and polysomes was consistently lower than that of similar preparations from liver. The concentrations of Na⁺ and K⁺ ions present during preparation had a great effect on the RNA/protein ratio. 6. Optimum conditions for the incorporation of amino acids into protein in vitro are reported. Cardiac ribosomes have a lower rate of incorporation of amino acids in vitro than liver ribosomes. 7. Heart cell sap is less active than liver cell sap: evidence is presented that a factor, present in liver cell sap and concerned with stimulating the synthesis of the peptide chain, is lacking in heart cell sap. 8. Pulse-labelling of perfused hearts followed by examination of the subcellular structures showed that the ribosomal fraction was the most active in the incorporation of amino acids in vitro.     

In vitro exchange of ribosomal subunits between free and membrane-bound ribosomes

Studies on the distribution of isotopieally labeled ribosomal subunits between free and membrane-bound ribosomes from rat liver showed that, upon release of nascent polypeptides in vitro, the small subunits of membrane-bound ribosomes could exchange with small subunits derived from free polysomes. However, under the same conditions, the large subunits of membrane-bound ribosomes did not exchange efficiently with large subunits derived either from free or bound polysomes; instead, the addition of large subunits caused a transfer of microsomal small subunits into a newly formed pool of free monomers.The small subunit exchange required a macromolecular fraction of the cell sap, was stimulated by ATP or GTP, and occurred at low concentrations of magnesium ions.Sodium dodecyl sulfate, polyacrylamide gel electrophoresis revealed close similarities between the protein complement of subunits from free and membrane-bound ribosomes, with the exception of one protein band which was more intense in free large subunits.     

The inhibition of protein synthesis in a rat brain system by ethionine in vitro and in vivo

Abstract— An amino acid incorporating system from rat brain has been used to study in vitro four aspects of protein synthesis: amino acid-AMP-enzyme complex formation; amino acid-tRNA synthesis; amino acid incorporation into protein and protein synthesis from presynthesized amino acid-tRNA. Ethionine (0.5 mm ) inhibited the system and the inhibition appeared to be in the formation of amino acid-tRNA. The inhibition in vitro was independent of the sex of the animal from which the system was derived. Pretreatment of animals in vivo with ethionine yielded in females only preparations deficient in incorporating capacity when tested in vitro. Exchange experiments demonstrated that the defect was in the pH 5 enzymes and not in the ribosomes. The inhibition in vitro was not reversed by addition of ATP and appeared to be competitive with the amino acid substrate.     

Cell-Free Protein Synthesis by Rumen Protozoa

SYNOPSIS. The characteristics of protein synthesis by cell-free extracts of mixed rumen protozoa have been investigated. ATP,¹ GTP, and an energy supply system were necessary for amino acid incorporation which was partially inhibited by cycloheximide but not by chloramphenicol (100 μg/ml). The system was particularly sensitive to the cation concentration of the incubation mixture, maximal incorporation requiring 5 mM Mg⁺⁺ and 50 mM K⁺ Incorporation was further stimulated by the addition of 0.25 mM spermidine or 0.25 mM MnCl₂. Sucrose gradient centrifugation of the cell sap after amino add incorporation showed that most of the incorporated radioactivity was associated with free polysomes. These polysomes contained 82 S ribosomes which dissociated in high Tris concentrations to yield 40 S and 55 S ribosomes.     

Protein Synthesis in Plant Microsomal System

An active microsomal system from 48-h germinating seeds of Vigna sinensis (L.) Savi has now been developed. It can incorporate amino acids into protein under both in vitro and in vivo conditions, provided dithiothreitol (a protective reagent for SH groups) and phenylthiourea (an inhibitor of phenol oxidase) are present in the buffer system for extraction; and provided the assay mixture contains added dithiothreitol. The system consists of microsomes or ribosomes, tRNA or pH 5 fraction and 20 natural amino acids, ATP and an ATP-generating system and GTP with requirement for Mg ions. The cell fractions possess aminoacyl-RNA synthetase activity as indicated by the aminoacylhydroxamate formation. Microsomal synthesis is stimulated by exogenous tRNA from Escheriehia coli or rat liver and sensitive to various inhibitors such as cyclo-heximide, chloramphenicol, fusidic acid. The ribosomal transfer reaction has absolute dependence on the microsomal wash, on the crude enzyme from the same participate source, and on a synthetic messenger. It is greatly suppressed by fusidic acid and by cycloheximide.     

Biochemical research on oogenesis: protein synthesis in whole cells and in cell-free extracts of Xenopus laevis immature ovaries

Nearly all tRNA molecules in previtellogenic oocytes of Xenopus laevis are included in nucleoprotein particles sedimenting at 42S. The tRNA-binding sites of these particles have several properties in common with those of the ribosomes. This suggests that the 42S particles might behave like unprogrammed ribosomes and be the site of a template-independent polymerization of amino acids. We expected this reaction to be insensitive to protein synthesis inhibitors, such as cycloheximide and puromycin. We found that these antibiotics almost completely inhibit the incorporation of labeled amino acids into protein, when added to the incubation medium of whole ovaries or free oocytes. In cell-free extracts of ovaries, the incorporation of amino acids is partially insensitive to cycloheximide and puromycin. When such extracts are fractionated by sucrose density centrifugation and incubated with ATP, a major peak of amino acid incorporation can be detected, which nearly coincides with the 42S particle peak.     

The Influence of Axis Removal on Protein Metabolism in Cotyledons of Pisum sativum L

The protein metabolism of cotyledons attached to the embryonic axis has been compared with that in cotyledons removed from the axis at the initiation of a 6-day imbibition. Total protein declined in the attached but not in the detached cotyledons. Concurrent with the decline in protein level in the intact cotyledons there was an increased capacity to incorporate exogenously supplied leucine into protein. In contrast, detached cotyledons showed a restricted capacity for protein synthesis. It was demonstrated that ribosomal preparations from cotyledons of intact seedlings contained an increasing proportion of polyribosomes as germination progressed and such ribosomes were active in in vitro amino acid incorporation. Ribosomal preparations from detached cotyledons contained few polyribosomes and had a restricted capacity to incorporate amino acids in vitro. The in vitro incorporation of phenylalanine was stimulated by polyuridylic acid with the stimulation being greatest in ribosomal preparations from detached cotyledons. The results suggest that an axis component may regulate the availability of messenger RNA in the cotyledons during germination.     

Interaction between glutathione and the endoplasmic reticulum in cyclic protein synthesis in sea urchin embryos.

Interaction between an oxidoreduction system and cyclic protein synthesis was studied in sea urchin embryos. When assayed enzymatically, in both in vivo and in vitro systems, the contents of GSH and GSSG varied inversely in a cyclic fashion. Diamide at 0.5 mM inhibited amino acid incorporation in not only the cyclic phase but also the basal phase, but 4-nitroquinoline-N-oxide at 1 μM inhibited only the cyclic phase. Sea urchin embryos contained membrane-bound ribosomes, and pulse-labeling with amino acids suggested that free ribosomes were responsible for the basal phase and membrane-bound ribosomes were responsible for the cyclic phase of amino acid incorporation. Thiol-disulfide interchanging enzyme was found in the endoplasmic reticulum fraction. An extract of the endoplasmic reticulm caused stimulation of binding of acetylphenylalanyl-tRNA to 40S ribosomes and polyphenylalanine synthesis in the presence of low GSH concentrations. An extract of the endoplasmic reticulum also catalyzed oxidoreduction from GSH to the KCl-soluble protein. Thus, the periodic stimulation of protein synthesis is interpreted to be the result of the periodic activation of membrane-bound ribosomes by the thiol-disulfide interchanging enzyme which accepts selectively the signal from the GSH cycle.     

Isolation and characterization of ribosomes from thoraces of the sheep blowfly,Lucilia cuprina,and their capacity for protein synthesis at different stages of development

The isolation and characterization of ribosomal fractions from thoraces of Lucilia cuprina have been described and their capacity for in vitro protein synthesis studied at different stages of development. There are large differences in the yield of ribosomes during development, and these probably reflect differences in the number of ribosomes in vivo. The amino acid incorporation activity varied at different stages of development and this variation correlated with the percentage of polysomes present. Maximal activity was found just after adult emergence. Results obtained by the use of supernatant fractions from different stages of development suggest that control of protein synthesis in this insect takes place at the level of translation.     

Events surrounding the early development of euglena chloroplasts: v. Control of paramylum degradation

The degradation of the storage carbohydrate, paramylum, is induced by light in wild-type Euglena gracilis Klebs var. bacillaris Pringsheim and in a mutant, W₃BUL, which lacks detectable plastid DNA. Treatment of wild type with cycloheximide in the dark produces 60% as much paramylum breakdown as light, whereas treatment with levulinic acid in the dark yields a slightly greater response than light. Both cycloheximide and levulinic acid produce a greater paramylum breakdown in the light than they do in the dark. Treatment of W₃BUL with levulinic acid in darkness produces a larger paramylum degradation than light, with values similar to wild type in the light. Treatment of W₃BUL with cycloheximide induces paramylum degradation in darkness, and as with wild type, light is slightly stimulatory in the presence of both cycloheximide or levulinic acid. Streptomycin brings about only a very small amount of paramylum breakdown in the dark and only slightly inhibits breakdown in the light. Thus paramylum breakdown induced by light does not require the synthesis of proteins on cytoplasmic or plastid ribosomes. A model which explains these results postulates the existence of a protein which inhibits paramylum breakdown. When the synthesis of this protein is prevented either by light, cycloheximide, or by levulinic acid acting as a regulatory analog of delta amino levulinic acid, paramylum breakdown takes place. Because levulinic acid is a better inducer than light in W₃BUL, W₃BUL may not be able to form as much delta amino levulinic acid in light as wild type. The small amount of induction by streptomycin is viewed as a secondary regulatory effect attributable to interference with plastid protein synthesis which affects regulatory signals from the plastid to the rest of the cell.     

Studies on the incorporation of ( 14 C)amino acids into protein by isolated rat brain nuclei

—Various parameters of the in vitro incorporation of [¹⁴C]amino acids into protein by cell nuclei isolated and purified from rat brain and liver were investigated. Nuclei purified through 2.2 m sucrose solution were capable of amino acid incorporation in vitro; and washing procedure to eliminate hypertonic sucrose before incubation was essential since sucrose in high concentration was inhibitory. Microbial contamination was found to be a serious source of error and the use of sterile conditions for incubation were necessary to obtain reproducible and valid results. Using completely sterile conditions, Na ⁺, K⁺, RNase, DNase, puromycin, cycloheximide and chloramphenicol were without any effect on the ability of brain and liver nuclei to incorporate labelled amino acids into protein. Results of time-course and preincubation experiments revealed that some factors essential for amino acid incorporation pass out of the nucleus into the medium. In addition, approximately 15 per cent of the labelled nuclear proteins with higher specific radioactivity was recovered in the incubation medium. Incorporation of [¹⁴C]leucine was proportional to the concentration of labelled amino acid and to the number of nuclei, and it is suggested that carefully controlled conditions of incubation are essential to obtain valid comparisons between different types of nuclei in terms of their relative abilities to incorporate amino acids in vitro. No evidence was obtained indicating isotope dilution phenomenon in these experiments. Whether or not in vitro incorporation of amino acid by nuclei represents protein synthesis is discussed.     

The influence of mitochondria on ribosomes. Cycloheximide resistance of ribosomes from petite mutants of saccharomyces cerevisiae

The influence of cycloheximide on amino acid incorporation into protein of standard strain ? + and cytoplasmic mutant ? ? of $\text{S.}\text{cerevisiae}$ was determined $\text{in}\text{vivo}$ and $\text{in}\text{vitro}$. $\text{In}\text{vivo}$ cycloheximide at the concentration which inhibits protein synthesis in ? + strain by over 60% has litle or no effect in mutant ? ? strain. $\text{In}\text{vitro}$ cycloheximide in the range of 0.05 to 0.3 ug/ml of incubation medium inhibits polyphenylalanine synthesis in ? + strain by 50% and in ? ? strain by less than 10%. Similar resistance to this antibiotic are shown in standard strain grown in anaerobic conditions. It has been found that the resistance to cycloheximide is associated with changes in cytoplasmic ribosomes and may depend on the integrity of mitochondrial system.     

Comparison of cell-free protein synthesis by different regions of chicken brain

The cell-free protein synthesis by the postmitochondrial supernatant from chicken cerebrum was twofold greater than protein synthesis by the cerebellum or optic lobes. Ribosomal aggregation of mRNA and ribonuclease activity of the postmitochondrial supernatant from the three brain regions was not statistically different. The higher protein synthetic activity of the cerebral postmitochondrial supernatant was associated with both the postribosomal supernatant (cell sap) and microsomal fractions. Cerebral monomeric ribosomes were more active in polyuridylic acid directed polyphenylalanine synthesis than monomeric ribosomes from either the cerebellum or optic lobes. The ability of cerebral cell sap to support polyuridylic acid directed polyphenylalanine synthesis was 1.6 to 2 times greater than cell sap from the other two regions. Cell sap factors other than tRNA^phe or phenylalanyl-tRNA synthetases appear to be responsible for the higher protein synthetic activity of the cbr cell sap.     

Stimulation of yeast mitochondrial protein synthesis by postpolysomal supernatants from yeast,rat liver,and Escherichia coli

Isolated yeast mitochondria incubated with a protein-synthesizing mixture containing excess oxidizable substrate, amino acids, MgCl₂, an ATP-regenerating system, and optimal levels of [³H]leucine cease protein synthesis after 30 min. Postpolysomal supernatants from either yeast, rat liver, or Escherichia coli can restore protein synthetic activity to depleted yeast mitochondria; however the addition of bovine serum albumin to the incubation mixture did not restore activity. The restored incorporation activity was sensitive to chloramphenicol, insensitive to cycloheximide, and proportional to the protein concentration of the supernatants. Furthermore, addition of all three high-speed supernatants to isolated mitochondria at time zero stimulated the rate of protein synthesis to a greater extent than when these fractions were added to depleted mitochondria. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate revealed that the translation products obtained from mitochondria labeled in vitro in the presence of supernatant fractions were identical to the proteins labeled by mitochondria in vivo; however, the synthesis of the bands corresponding to subunit III of cytochrome oxidase, cytochrome b, and VAR-3 was stimulated to the greatest extent. The stimulatory activity in the supernatants was non-dialyzable, insensitive to treatment with ribonuclease A, but completely abolished by pretreatment with trypsin suggesting that the stimulatory factor(s) is of a protein nature. The postpolysomal supernatants did not incorporate amino acids into protein when incubated without mitochondria. These results suggest that the protein synthetic capacity of mitochondria is apparently limited by extramitochondrial proteins which are present in either yeast, rat liver, or E. coli.     

The breakdown of Tetrahymena ribosomes by lysosomal enzymes: Inhibition by cytosol

• 1.1. Extracts from Tetrahymena lysosomes contained acid RNase and proteinase. At pH 7.4 there was appreciable proteinase activity which was inhibited by a heat-stable protein present in cell sap.
• 2.2. Lysosomal enzymes rapidly converted 80S ribosomes to subunits at pH 7.4. Hydrolysis of ribosomal RNA was very slow at pH 7.4 but rapid at pH 5.0.
• 3.3. These reactions were inhibited by proteinase inhibitors and by cell sap, but the latter was relatively ineffective at pH 5.0.
• 4.4. It seems unlikely that ribosome breakdown in vivo is initiated by the release of lysosomal enzymes into the cytosol.

     

Synthesis of macromolecules during microcyst germination in the cellular slime mold Polysphondylium pallidum

Microcyst germination in the cellular slime mold Polysphondylium pallidum is a useful model for studying macromolecular changes necessary for or coincident with the transition from one cell type (cyst) to another (amoebae). Protein synthesis starts soon after cysts are incubated under permissive conditions, as evidenced by the incorporation of precursors and the appearance of polysomes. Sodium dodecyl sulfate-polyacrylamide gel analysis of proteins made at intervals during germination shows that protein synthesis is developmentally regulated during this process. RNA synthesis also begins early during germination. Cysts contain polyadenylated RNA that can stimulate the incorporation of radioactive amino acids into protein in an in vitro wheat germ protein synthesizing system. The concentration of poly(A)-containing RNA increases during germination and during inhibition of protein synthesis by cycloheximide.     

Interactions of liver encoplasmic reticulum membranes and polysomes in vitro.

The interactions of various preparations of endoplasmic reticulum membranes and polysomes have been studied by means of a sandwich sucrose gradient that clearly isolates free ribosomes, smooth endoplasmic reticulum (S.E.R.) and rough endoplasmic reticulum (R.E.R.) from the microsomal fraction of rat liver homogenates. Reconstructed rough membranes separate well from the native R.E.R. but occupy the same position along the gradient as the S.E.R. and the rough membranes, stripped of their ribosomes by means of LiCl. Native R.E.R. and S.E.R. do not bind any added labeled polysomes at 0 degree C; previous treatment with LiCl does not modify the behavior of S.E.R. The presence of cell sap during the binding reaction does not increase polysome fixation by stripped-rough membranes but protects in some way the polysomes and preserves all their original functional capacity of amino acid incorporation into protein.     

Translation of brain messenger ribonucleic acids in homologous,heterologous and mixed cell free systems

Optimum conditions were determined for translation of rat brain messenger RNA in vitro using three heterologous systems (wheat germ, Krebs ascites cell and reticulocyte) and a homologous system containing ribosomal subunits and factors from brain. The four systems showed similarities, as well as differences, in regard to their requirements. Although spermine partially replaced magnesium ions in all the four, it stimulated protein synthesis in the extracts of reticulocyte and wheat germ, but not in those of ascites cell or brain. When potassium ions were added as acetate instead of chloride, amino acid incorporation was enhanced and the optimum was shifted to much higher concentrations of potassium (110–120 mM) than was observed with KCl (80 mM). These differences were probably due to inhibition by high concentrations of chloride when KCl was used as the sole source of potassium.Under optimum conditions for each system, translation of brain messenger RNA in the brain system was inferior to the other three extracts, when based on equivalent amounts of ribosomes present in the reaction mixture. However, the homologous system was able to sustain linear incorporation of amino acid for a much longer period than the others, indicating that homologous factors may play a role in the translation of brain messenger RNA.