Effect of nucleoside triphosphate and magnesium ion concentration on the stability and function of rat liver polysomes in vitro

The effects of different concentrations of ATP, GTP, UTP and CTP on polysome stability and function in a cell-free protein-synthesizing system prepared from rat liver were studied. Increasing the concentration of ATP in the incubation medium to 15mm resulted in progressive disaggregation of the polysomes; at ATP concentrations above 2mm their capacity to incorporate amino acids into peptide chains diminished. The same disaggregation phenomenon could be produced by incubating polysomes in a buffered medium containing 5mm-Mg(2+) and increasing concentrations of ATP. Although the disaggregating action of ATP could be prevented by increasing Mg(2+) concentration, the amino acid incorporation in the cell-free protein-synthesizing system remained impaired. The effects of different concentrations of GTP, UTP and CTP on polysome stability were similar to those of ATP. Increasing the concentrations of each nucleoside triphosphate also inhibited the hydrolysis of GTP in the cell-free protein-synthesizing system.     

Regulation of polyribosome formation and protein synthesis in the uterus. Isolation of cytoplasmic ribonucleoprotein particles and the principal properties of the cell-free protein-synthesizing system

1. Three procedures for isolating ribonucleoprotein particles from the cytoplasmic fraction of rat-uterus homogenates are described. By procedure 1, ribonucleoprotein particles were isolated in the presence of 5mm-Mg(2+) and 25mm-K(+), and the postmitochondrial supernatant fraction was made to 1.3% (w/v) in potassium deoxycholate. About 50% of the RNA and protein of the microsomal fraction was recovered in the monomeric ribosomes isolated. By procedure 2, ribonucleoprotein particles were isolated in the presence of 10mm-Mg(2+) and 0.1m-K(+), and in the absence of detergent. The ribosomes obtained were primarily polymeric, but recovery of microsomal RNA and protein was only 32%. By procedure 3, ribonucleoprotein particles were isolated according to procedure 1 but without the use of detergent. A mixture of polymeric and monomeric ribosomes was obtained, and the recovery of microsomal RNA and protein was about 60%. 2. Uterine polymeric and monomeric ribosomes, isolated by procedure 3 and designated ;polyribosomal preparation', were examined for protein-synthesizing capabilities. The principal properties of the cell-free protein-synthesizing system containing the polyribosomal preparation are described. The efficiency of amino acid incorporation in the complete system incubated for 30min. and containing the polyribosomal preparation was found to be either 2.5 molecules of [(14)C]leucine or 2.2 molecules of [(14)C]-valine incorporated/ribosome. Assay of the preparation in the complete cell-free system containing 10mm-sodium fluoride indicated that 40% of the incorporation activity is a result of initiation of new polypeptide chains and 60% is due to completion of previously existing chains. Monomeric ribosomes obtained by various treatments of the polyribosomal preparation with sodium fluoride, ribonuclease and potassium deoxycholate had decreased incorporation activity in the cell-free system. However, monomeric ribosomes obtained by treatment with sodium fluoride only had an incorporation activity 50% greater than that of monomers obtained by treatment with ribonuclease only. 3. The results indicate that uterine polymeric and monomeric ribosomes are sites of amino acid incorporation in vivo and in vitro. It is concluded that most polymeric and monomeric ribosomes occurring in the cytoplasmic fraction of the uterus are free and unattached to membranes, and that the polyribosomes are relatively unstable.     

Dynamics of the biosynthesis of components of the protein synthesizing apparatus of the rat liver at the stage of restoration of translation, inhibited by cycloheximide

The biosynthesis of proteins, ribosomal RNA and other components of the rat liver protein-synthesizing system during the reparation and subsequent activation of translation inhibited by a sublethal dose cycloheximide (CHI, 3 mg/kg) was studied. It was found that the incorporation of labeled precursors into proteins and ribosomal rRNA isolated from free and membrane-bound polysomes is repaired already 3 hours after CHI injection. 6-9 hours thereafter, the level of component labeling reaches control values, whereas the total protein biosynthesis is retarded. After 12-24 hours, marked stimulation of ribosome biosynthesis and the integration of ribosomes into polysomes are observed together with an asymmetric accumulation of excessive amounts of newly synthesized 40S subunits into polysomes 12 hours after CHI infection. The putative mechanisms of the activation of expression of the part of the genome responsible for protein and ribosomal rRNA synthesis as well as for the synthesis of other components of the protein-synthesizing system are discussed.     

In vitro incorporation of labelled amino acids into heart muscle ribosomes at early and late stages of compensatory heart hyperfunction]

The activity of a protein-synthesizing cell-free system from heart muscle was studied at early and late stages of compensatory heart hyperfunction. It was found that the incorporation of amino acids into heart ribosomes during 48 hours after the hyperfunction had been produced, increased by 30% as compared to the control. The incorporation of amino acids into heart ribosomes at the late stage of hyperfunction (after 6 months) was decreased by 46% as compared to the early stages. The addition of homologous tRNA to the cell-free system of protein synthesis under prolonged heart hyperfunction stimulated the incorporation of amino acids into the ribosomes by 40--50%.     

Isolation and Characterization of Collagen - Synthesizing Polysomes from Chick Embryos

Collagen - synthesizing polysomes were isolated by low-speed oentrifugation of the post-mitochomirial supernatant of chick homogenates. Electron microscopy of the fraction thus isolated shows it to be exclusively composed of ribosomes. Amino acid incorporation in vitro showed that these particles were efficient in the incorporation of proline, but not tryptophan, as opposed to ribosomes obtained from the supernatant of the low-speed oentrifugation. The incorporation process was highly dependent on GTP, and exibited an optimal Mg²⁺ concentration of 5.6 mN. The reaction was inhibited by RNase, elongation inhibitors as anysomycin, sparsomycin, fusidic acid and GDPCP. It was also moderately inhibited by initiation inhibitors such as aurintricarboxilic acid and pyrocatechol violet. The product of the incorporation was characterized as collagen by its sensitivity towards purified collagenase, lack of tryptophan, chromatography in CN-cellulose and molecular sieve chromatography in Sephadex G-200.     

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.     

Isolation and characterization of collagen-synthesizing polysomes from chick embryos.

Collagen-synthesizing polysomes were isolated by low-speed centrifugation of the post-mitochondrial supernatant of chick homogenates. Electron microscopy of the fraction thus isolated shows it to be exclusively composed of ribosomes. Amino acid incorporation in vitro showed that these particles were efficient in the incorporation of proline, but not tryptophan, as opposed to ribosomes obtained from the supernatant of the low-speed centrifugation. The incorporation process was highly dependent on GTP, and exibited an optimal Mg2+concentration of 5.6mM. The reaction was inhibited by RNase, elongation inhibitors as anysomycin, sparsomycin, fusidic acid and GDPCP. It was also moderately inhibited by initiation inhibitors such as aurintricarboxilic acid and pyrocatechol violet. The product of the incorporation was characterized as collagen by its sensitivity towards purified collagenase, lack of tryptophan, chromatography in CM-cellulose and molecular sieve chromatography in Sephadex G-200.     

Action of x-ray irradiation on the nuclear envelope of rat liver cells]

X-irradiation of isolated nuclear envelopes (NE) has revealed their high radiosensitivity, while irradiation of isolated intact nuclei in vitro, in the doses up to 5000 r 18--20 hours after partial hepatectomy, produced no morphological changes in NE. The damaging effect of irradiation on both nuclei and mitochondria (Mt) was revealed only with a decrease in cytochrome-c-oxidase (CO) activity in parallel with an increase in the radiation dose. One hour after the whole body irradiation of rats in the beginning of S-period, the damaging effect was recorded in both NE and Mt at the doses of 50 and 150 t, and was enhanced with the increase of irradiation dose. Morphological changes were observed mostly in the outer nuclear membrane, which lost its distinct outline and disappeared from some nuclear regions. Lethal radiation doses produced a decrease in the number of pore complexes (PC) with their evident segregation from the membranes. After irradiation in a dose of 1200 r, only the residue or "ghosts" of the PCs remained. After irradiation in doses up to 400 r, the CO-activity recovered during the first hour in Mt and during first two hours in the nuclei.     

Cell-free Synthesis of Globulin by Developing Oat (Avena sativa L.) Seeds

The primary storage protein synthesized during oat (Avena sativa L.) groat development is a globulin. Polysomes were isolated from oat groats 12 days after anthesis. These polysomes directed the incorporation of radioactive amino acids into protein in a cell-free protein synthesis system containing wheat germ supernatant. The Mg(2+) optimum was 4 mm, the pH optimum was 6-8, and the amount of amino acid incorporation depended on polysome concentration. Incorporation of amino acids was linear for about 10 min and approached a maximum after 20 min. Using the initiation inhibitor, T-2 toxin, it was determined that about 36% of the amino acid incorporation was due to the initiation of new polypeptide chains. The in vitro product co-electrophoresed with authentic oat groat globulin on polyacrylamide-sodium dodecyl sulfate (SDS) gels. The cyanogen bromide peptides of the in vitro product partially corresponded with those from authentic globulin when electrophoresed on polyacrylamide-SDS gels. These data suggest that the in vitro product is primarily oat globulin. The polysome population was separated into membrane-bound and free polysomes. Membrane-bound polysomes synthesized about twice the amount of protein as did free polysomes. Products synthesized in vitro on both types of polysomes were essentially the same.     

Partial purification, characterization and translation in vitro of rat liver metallothionein messenger ribonucleic acid.

Poly(A)+ (polyadenylated) mRNA coding for metallothioneins was purified 13-fold from rat liver polyribosomes and was identified by its ability to direct the biosynthesis of these proteins in a wheat-germ cell-free system. The carboxymethylated products of the protein-synthesizing system in vitro were analysed with sodium dodecyl sulphate/20% polyacrylamide-gel electrophoresis. The labelled compounds [3H]serine and [35S]cysteine were incorporated at high specific radioactivity into proteins that co-migrated with authentic metallothioneins. No [3H]leucine incorporation was found, in agreement with the amino acid composition of the metallothioneins. Metallothionein mRNA had a sedimentation coefficient of 9 S and carried a maximum of four ribosomes. At 5 h after a subcutaneous injection of ZnCl2 or CdCl2 (10 mumol/kg body wt.), the amount of this mRNA increased approx. 2- and 4-fold respectively, on the basis of translation in vitro. The increase in metallothionein mRNA (defined by translation in the wheat-germ system) was transient and, after CdCl2 treatment, fell back to control values by 17 h. Metallothioneins constituted a maximum of 0.8% of the total protein products synthesized in the wheat-germ system by total mRNA isolated from rat liver after CdCl2 treatment.     

In vitro amino acid incorporation into myosin by free polysomes of rat skeletal muscle

Rat skeletal muscle polysomes were separated into free and membrane-bound fractions by centrifugation through 2M sucrose. About 80% of total ribosomes extracted were recovered as free polysomes. Sucrose gradient experiments showed similar size distribution patterns for both free and bound polysomes. Chromatographic and electrophoretic analyses of proteins in the cell free amino acid incorporation system indicated that free polysomes are capable of synthesizing myosin.     

Reduced ribosomal binding of eukaryotic elongation factor 2 following ADP-ribosylation. Difference in binding selectivity between polyribosomes and reconstituted monoribosomes

The biological activity of elongation factor 2 (EF-2) following NAD+ - and diphtheria-toxin-dependent ADP-ribosylation was studied (i) in translation experiments using the reticulocyte lysate system and (ii) in ribosomal binding experiments using either reconstituted empty rat liver ribosomes or programmed reticulocyte polysomes. Treatment of the lysates with toxin and NAD+ at a NAD+/ribosome ratio of 4 resulted in a 90% inhibition of the amino acid incorporation rate. The inhibition was overcome by the addition of native EF-2. At this level of inhibition more than 90% of the EF-2 present in the lysates was ADP-ribosylated and the total ribosome association of EF-2 was reduced by approx. 50%. All of the remaining unmodified factor molecules were associated with the ribosomes, whereas only about 3% of the ribosylated factor was ribosome-associated. The nucleotide requirement for the binding of EF-2 to empty reconstituted rat liver ribosomes and programmed reticulocyte polysomes was studied together with the stability of the resulting EF-2 X ribosome complexes using purified 125I-labelled rat liver EF-2. With both types of ribosomes, the complex formation was strictly nucleotide-dependent. Stable, high-affinity complexes were formed in the presence of the non-hydrolysable GTP analogue guanosine 5'-(beta, gamma-methylene)triphosphate (GuoPP[CH2]P). In contrast to the reconstituted ribosomes, GTP stimulated the formation of high-affinity complexes in the presence of polysomes, albeit at a lower efficiency than GuoPP[CH2]P. The formation of high-affinity complexes was restricted to polysomes in the pretranslocation phase of the elongation cycle. Low-affinity post-translocation complexes, demonstrable after fixation, were formed in the presence of GTP, GuoPP[CH2]P and GDP. In polysomes, these complexes involved a different population of particles than did the high-affinity complexes. In the binding experiments using reconstituted or programmed ribosomes, the pretranslocation binding of EF-2 observed in the presence of GuoPP[CH2]P was reduced by approx. 50% after ADP-ribosylation, whereas the post-translocation binding in the presence of GDP was unaltered. The data indicate that the inhibition of translocation caused by diphtheria toxin and NAD+ is mediated through a reduced affinity of the ADP-ribosylated EF-2 for binding to ribosomes in the pretranslocation state.     

Controlled proteolysis of nascent polypeptides in rat liver cell fractions. II. Location of the polypeptides in rough microsomes

Rough microsomes were incubated in an in vitro amino acid-incorporating system for labeling the nascent polypeptide chains on the membrane-bound ribosomes. Sucrose density gradient analysis showed that ribosomes did not detach from the membranes during incorporation in vitro. Trypsin and chymotrypsin treatment of microsomes at 0° led to the detachment of ribosomes from the membranes; furthermore, trypsin produced the dissociation of released, messenger RNA-free ribosomes into subunits. Electron microscopic observations indicated that the membranes remained as closed vesicles. In contrast to the situation with free polysomes, nascent chains contained in rough microsomes were extensively protected from proteolytic attach. By separating the microsomal membranes from the released subunits after proteolysis, it was found that nascent chains are split into two size classes of fragments when the ribosomes are detached. These were shown by column chromatography on Sephadex G-50 to be: (a) small (39 amino acid residues) ribosome-associated fragments and (b) a mixture of larger membrane-associated fragments excluded from the column. The small fragments correspond to the carboxy-terminal segments which are protected by the large subunits of free polysomes. The larger fragments associated with the microsomal membranes depend for their protection on membrane integrity. These fragments are completely digested if the microsomes are subjected to proteolysis in the presence of detergents. These results indicate that when the nascent polypeptides growing in the large subunits of membrane-bound ribosomes emerge from the ribosomes they enter directly into a close association with the microsomal membrane.     

Microsomal components in relation to amino acid incorporation by preparations from the developing rat brain

1. After incorporation of [(14)C]valine in vitro, cerebral microsomes were separated into membrane-bound and free ribosomes by sucrose-density-gradient centrifugation. 2. In preparations from both 4-day-old and adult rats, free and bound ribosomes incorporated [(14)C]valine. Free ribosomes could be found as polysomes, which were highly active. 3. Microsomes labelled with [(14)C]valine in vitro were fractionated after deoxycholate treatment into a preliminary sediment, sedimented at 105000g (5min.), and ribonucleoprotein particles, sedimented at 150000g (70min.), to determine the role of membrane-bound ribosomes. In the adult the ribonucleoprotein particles retained most of the radioactivity, whereas in the young the preliminary sediment was as highly labelled as the ribonucleoprotein particles. 4. The labelled preliminary sediment from young preparations was both ribonuclease- and deoxycholate-resistant, and the nature of this material is discussed in terms of a possible structural component of microsomal membranes.     

Regulation of polyribosome formation and protein synthesis in the uterus. Effect of ovariectomy and administration of oestradiol-17beta on the amino acid-incorporation activity in vitro and the cytoplasmic concentration in vivo of polyribosomal preparation

1. Three procedures for isolating ribonucleoprotein particles from the cytoplasmic fraction of rat-uterus homogenates are described. By procedure 1, ribonucleoprotein particles were isolated in the presence of 5mm-Mg²⁺ and 25mm-K⁺, and the postmitochondrial supernatant fraction was made to 1·3% (w/v) in potassium deoxycholate. About 50% of the RNA and protein of the microsomal fraction was recovered in the monomeric ribosomes isolated. By procedure 2, ribonucleoprotein particles were isolated in the presence of 10mm-Mg²⁺ and 0·1m-K⁺, and in the absence of detergent. The ribosomes obtained were primarily polymeric, but recovery of microsomal RNA and protein was only 32%. By procedure 3, ribonucleoprotein particles were isolated according to procedure 1 but without the use of detergent. A mixture of polymeric and monomeric ribosomes was obtained, and the recovery of microsomal RNA and protein was about 60%. 2. Uterine polymeric and monomeric ribosomes, isolated by procedure 3 and designated `polyribosomal preparation', were examined for protein-synthesizing capabilities. The principal properties of the cell-free protein-synthesizing system containing the polyribosomal preparation are described. The efficiency of amino acid incorporation in the complete system incubated for 30min. and containing the polyribosomal preparation was found to be either 2·5 molecules of [¹⁴C]leucine or 2·2 molecules of [¹⁴C]-valine incorporated/ribosome. Assay of the preparation in the complete cell-free system containing 10mm-sodium fluoride indicated that 40% of the incorporation activity is a result of initiation of new polypeptide chains and 60% is due to completion of previously existing chains. Monomeric ribosomes obtained by various treatments of the polyribosomal preparation with sodium fluoride, ribonuclease and potassium deoxycholate had decreased incorporation activity in the cell-free system. However, monomeric ribosomes obtained by treatment with sodium fluoride only had an incorporation activity 50% greater than that of monomers obtained by treatment with ribonuclease only. 3. The results indicate that uterine polymeric and monomeric ribosomes are sites of amino acid incorporation in vivo and in vitro. It is concluded that most polymeric and monomeric ribosomes occurring in the cytoplasmic fraction of the uterus are free and unattached to membranes, and that the polyribosomes are relatively unstable.     

The distribution of ribosomes between different functional states in livers of fed and starved mice

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 ³H or ¹⁴C 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.     

If bystander effects for apoptosis occur in spleen after low-dose irradiation in vivo then the magnitude of the effect falls within the range of normal homeostatic apoptosis

To test whether bystander effects occur in vivo after low doses of radiation relevant to occupational and population exposure, we exposed mice to whole-body X-radiation doses (0.01 and 1 mGy) where only a proportion of cells would receive an electron track. We used a precise method to analyze the apoptosis frequency in situ in spleen tissue sections at 7 h and 1, 3 and 7 days after irradiation to determine whether an increase in apoptosis above that predicted by direct effects was observed. No significant changes in the apoptosis frequency at any time after low-dose irradiation were detected. Apoptosis was induced above endogenous levels by five- to sevenfold 7 h after 1000 mGy. Using these data, the expected increases in apoptosis 7 h after a dose of 1 mGy or 0.01 mGy were calculated based on the assumption that induction of apoptosis would decrease linearly with dose. The magnitude of potential bystander effects for apoptosis that could be detected above homeostatic levels after these low doses of radiation was determined. A substantial bystander effect for apoptosis (>50-fold above direct effects) would be required before such proposed effects would be identified using 10 animals/treatment group as studied here. These data demonstrate that amplification of apoptosis even due to a substantial bystander effect would fall within the homeostatic range.     

Protein Synthesis in a Cell-Free Extract from Staphylococcus aureus

Cell-free Staphylococcus aureus extracts have been prepared which actively incorporate amino acids into protein. The requirements for amino acid incorporation of this preparation were strongly suggestive of de novo protein synthesis, since it showed an absolute requirement for ribosomes, 105,000 × g supernatant fluid, energy source, and magnesium ion. The stability of these extracts was greatly improved by use of dithiothreitol instead of mercaptoethanol as a sulfhydryl protecting reagent. Data were presented to show that the binding of aminoacyl-soluble ribonucleic acid to ribosomes did not require guanosine triphosphate and supernatant enzyme. The major characteristic which distinguishes this system from other cell-free systems is the much higher magnesium concentration required to maintain ribosomes intact and to obtain the maximal incorporation of amino acids. Addition of polyuridylic acid, polyadenylic acid, or polycytidylic acid caused about 60-fold, 30-fold, or 4-fold stimulation of the incorporation of phenylalanine, lysine, or proline, respectively. Studies by density gradient sedimentation indicated that radioactive polyuridylic acid or polyadenylic acid was associated with the monosomes. This complex can actively synthesize polypeptides. On the other hand, the nascent protein synthesized under the direction of endogenous messenger ribonucleic acid was associated with both polysomes and monosomes.     

The effect of chick-liver ribonucleic acid in amino acid-incorporation systems from rat liver

1. Rat-liver microsomes, ribonucleoprotein particles and a fraction mainly consisting of microsomal membranes were tested for their ability to incorporate amino acids into protein in the presence of ATP, GTP, phosphoenolpyruvate and pyruvate kinase. Addition of polyuridylic acid or of ribonucleic acid from rat-liver nuclei stimulated the incorporating activities. 2. These protein-synthesizing systems were found to be susceptible to ribonucleic acid from chick-liver nuclei as well. The biological activity of the ribonucleic acid from chick liver was measured by its capacity to stimulate amino acid incorporation. 3. In the presence of chick-liver ribonucleic acid, the ribonucleoprotein particles from rat liver showed an increased radioactivity in ribosomal units with a sedimentation constant higher than 70s. 4. This was investigated by sucrose-gradient centrifugation or by column chromatography on agarose suspensions.