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.     

Intracellular distribution of neutral proteinases and inhibitors in pig leucocytes. Isolation of two inhibitors of neutral proteinases.

Granule and post-granular-supernatant fractions were obtained from pig leucocyte cells by differential centrifugation in 0.34 M sucrose. Granule extract possesses proteinase activity at acid and at neutral pH. Three groups of neutral and a group of acid proteinases were isolated from granule extracts by chromatography on DEAE-cellulose. In the first group are present elastase-like and plasminogen-activator proteinases, that are inhibited by diisopropylphosphorofluoridate, alpha1-antitrypsin, intracellular leucocyte inhibitor and partly with p-aminomethylbenzoic acid and Trasylol. The second group of neutral proteinases is unstable under the conditions of isolation used the third group of neutral proteinases comprises collagenases that are inhibited by ethylenediamine tetraacetic acid disodium salt, alpha1-antitrypsin and leucocyte inhibitor. The acid proteinases are inhibited only with pepstatin, up to 90%. In the post-granular supernatant was found the acid proteinase activity towards hemoglobin and casein, and non-stable neutral proteolytic activity towards bovine serum albumin and serum gamma globulin. In the post-granular supernatant also the inhibitors of neutral proteinases were found. By gel filtration on Sephadex G-100 and ion-exchange chromatography on CM-cellulose two inhibitors of neutral proteinases were isolated. The majority of the inhibitor capacity (about 80%) of post-granular supernatant was eluted together with ovalbumin (Mr 43000) and the remainder with cytochrome c (12300). These inhibitors inhibit the granule neutral proteinases, acting on all substrates used, but do not inhibit granule acid proteinase. Inhibition effects of post-granular-supernatant inhibitors on trypsin and chymotrypsin were obtained only when bovine serum albumin was used as substrate. Inhibitors of post-granular supernatant are stable at pH 6-8, but unstable in the pH rnage 2-5 and are thermolabile.     

ISOLATION OF CYTOPLASMIC AND CHLOROPLAST RIBOSOMES AND THEIR DISSOCIATION INTO ACTIVE SUBUNITS FROM CHLAMYDOMONAS REINHARDTII

A mixture of cytoplasmic (80S) and chloroplast (70S) ribosomes from Chlamydomonas reinhardtii was freed of contaminating membranes by sedimentation of the postmitochondrial supernatant through a layer of 1.87 M sucrose. The purified ribosomes were separated into 80S and 70S fractions by centrifugation at a relatively low speed on a 10–40% sucrose gradient containing 25 mM KCl and 5 mM MgCl₂. Both the 80S and 70S ribosomes were dissociated into compact subunits by centrifugations in 5–20% high-salt sucrose gradients. The dissociations of both ribosomal species under these conditions were not affected by the addition of puromycin, indicating that the ribosomes as isolated were devoid of nascent chains. Subunits derived from the 80S ribosomes had apparent sedimentation coefficients of 57S and 37S whereas those from the 70S ribosomes had apparent sedimentation coefficients of 50S and 33S. In the presence of polyuridylic acid and cofactors, the 80S and 70S ribosomes incorporated [¹⁴C]phenylalanine into material insoluble in hot TCA. The requirements for incorporation were found to be similar to those described for eukaryotic and prokaryotic ribosomes. Experiments with antibiotics showed that the activity of the 80S ribosomes was sensitive to cycloheximide, whereas that of the 70S ribosomes was inhibited by streptomycin. The isolated subunits, when mixed together in an incorporation medium, were also active in the polymerization of phenylalanine in vitro.     

Regulation of Protein Synthesis in Zoospores of Blastocladiella

The factors responsible for the regulation of protein synthesis in the zoospores of Blastocladiella emersonii were studied by means of cell fractionation and in vitro assays. Charged transfer ribonucleic acid (tRNA) and aminoacyl-tRNA synthetases were found both inside the membrane-bound, ribosomal nuclear cap, and in the extracap cytoplasm. Ribosomes isolated from zoospore nuclear caps in low salt buffer failed to support polyuridylic acid-dependent phenylalanine incorporation. After washing with high salt buffer, the cap ribosomes were equivalent in activity to similarly prepared plant ribosomes. Both the high-salt wash from cap ribosomes and the extracap supernatant fraction contained an unidentified material which inhibited aminoacyl-tRNA binding and peptide bond formation by ribosomes. Ribosomal binding of polyuridylic acid was not inhibited. Washed cap ribosomes supported very low incorporation rates without added messenger RNA, and were highly dependent upon added poly U for phenylalanine incorporation, indicating a low level of messenger in nuclear caps. It is concluded that enclosure of the ribosomes in the nuclear cap does not in itself prevent protein synthesis, and that the lack of activity may be due to the presence of a ribosome inhibitor.     

RIBOSOMAL SUBUNITS FROM NEONATAL MOUSE BRAIN HIGHLY ACTIVE IN POLYPHENYLALANINE SYNTHESIS

Abstract— A highly active subcellular protein synthesising system is described, in which uncomplexed ribosomes isolated from 5 to 7 day old mouse brain can be reprogrammed with polyuridylic acid. Either purified free polyribosomes or microsomes were used as the starting material for the preparation of uncomplexed ribosomes by treatment with 0.5 m -KCl and puromycin. After reduction of the salt concentration 80S ribosomes were isolated by washing through sucrose. When, subsequently, zonal centrifugation in equivolumetric sucrose gradients containing 0.5 m -KCI was performed, purified ribosomal subunits were obtained. Cross-contamination of subunits was less than 5%. Re-associated ribosomes and recombined isolated ribosomal subunits both showed high activities in vitro. Incorporation levels of 50–60 phenylalanine residues per ribosome could be reached, at a rate of 0.5–2.0 residues/min/ribosome, depending on the activity of the high speed supernatant enzymes added. It was shown by paper chromatography of the cell-free product that only oligophenylalanine formation takes place. It was estimated that 6&70% of the ribosomes present in vitro were actively participating in the protein synthesis process.     

CRF-stimulated biosynthesis of ACTH in a cell-free system from rat anterior pituitaries

A cell-free system consisting of ribosomes, pH 5 enzymes and supernatant prepared from rat anterior pituitaries was found to be active in the incorporation of 3H-serine into ACTH. The rate of biosyntesis of ACTH, in a cell-free system as, measured by the incorporation of radioactive amino acid, and the rate of biological activity were markedly increased by the addition of CRF. The synthesis of ACTH was significantly inhibited by puromycin and RNAase but was not significantly inhibited by actinomycin D and DNAase.     

Properties of 5-fluorouracil-containing ribonucleic acid and ribosomes from Bacillus subtilis

Growth of a strain of Bacillus subtilis that requires uracil, thymine, adenine, and tryptophan in the presence of 5-fluorouracil (FU) results in the synthesis of ribonucleic acid (RNA) and ribosomes in which 55 to 65% of the RNA uracil has been replaced by the fluorine derivative. Examination of analogue-containing ribosomes by sucrose density gradient centrifugation and thermal denaturation studies suggests that, as far as the size, shape, and packing structure are concerned, extensive FU substitution has little or no effect. FU appears to replace uracil in RNA without selectivity for one RNA class over another, as determined by methylated albumin-kieselguhr column chromatography and sucrose density gradient centrifugation. The total amino acid content of the cells is markedly affected by growth in the presence of FU. The possibility of an FU effect on genetic translation is discussed.     

Effect of ribosome treatment on sensitivity to ricin A chain

Rat liver ribosomes treated with catalytic amounts (30 ng/ml) of ricin A chain are inhibited about 80% when assayed immediately. However, the same ribosomes assayed after separation from A chain by centrifugation have partially recovered their activity in the translation of polyuridylic acid. The extent of recovery is dependent on magnesium ion concentration. Even though the activity of A chain-treated ribosomes is increased after centrifugation, they are not sensitive to further treatment with ricin A chain. Except for impure ribosomes, isolated by centrifugation of crude homogenate, the overall sensitivity of ribosomes after different treatments was the same.     

Cell-free amino acid-incorporating system from Pseudomonas indigofera

Shiio, Tsuru (Washington State University, Pullman), and Bruce A. McFadden. Cell-free amino acid-incorporating system from Pseudomonas indigofera. J. Bacteriol. 90:978-983. 1965.-A cell-free preparation from Pseudomonas indigofera incorporated C(14)-phenylalanine and C(14)-leucine into a product which was insoluble in hot trichloroacetic acid. The phenylalanine incorporation process, which had a temperature optimum of 30 C and a pH optimum of 7.6, had many characteristics of protein synthesis. The process depended upon both "ribosomes" and supernatant fraction from centrifugation at 105,000 x g. Incorporation required adenosine triphosphate, apparently depended upon guanosine triphosphate, and was inhibited by chloramphenicol, puromycin, actinomycin, ribonuclease, and deoxyribonuclease. Leucine incorporation was also studied and had many similar characteristics. C(14)-phenylalanine uptake was stimulated by sRNA or polyuridylic acid, and together these substances had a synergistic effect upon stimulation. The incorporation of C(14)-phenylalanine into a product which was precipitated by antiserum to crystalline isocitrate lyase was also observed.     

Incorporation of orally administered radioactive precursors into nucleic acids and ribosomes of housefly ovaries

After emergence female houseflies were fed for 4 days on a diet containing ¹⁴C-orotic acid and ³H-thymidine-5-triphosphate, or ³H-leucine. Nucleic acids and ribosomes were then isolated from the ovaries and studied by MAK column chromatography and sedimentation analysis respectively. The ultraviolet absorption and radioactivity of the fractions were also measured. After MAK column chromatography, the u.v. elution pattern showed that only tow distinct peaks, corresponding to tRNA and rRNA were present. A similar elution pattern was obtained by measuring the ¹⁴C from ¹⁴C-orotic acid incorporated into the RNA. Because of the small quantity present, DNA was not measurable by u.v. absorption, but by determining the incorporation of ³H from ³H-TTP, its presence was clearly evident.Sedimentation analysis of ovarian ribosomes revealed four polymeric forms besides subunits and monomers. The incorporation of ¹⁴C-orotic acid and ³H-leucine into the ribosomes was used to follow the synthesis of rRNA and rProtein respectively. Sucrose density gradient centrifugation of the rRNA indicated that the ovarian rRNA consisted primarily of 28 and 18 S particles.     

Protein synthesis in tomato-fruit locule tissue: Incorporation of amino acids into protein by aseptic cell-free systems

1. Osmotically disrupted protoplasts and isolated plastids from tomato-fruit locule tissue were found capable of incorporating (14)C-labelled amino acids under aseptic conditions into an exhaustively washed trichloroacetic acid-insoluble protein fraction. 2. The disrupted protoplast system incorporated 20-45mumumoles of amino acid/mg. of protein in 10min. The isolated plastid system incorporated 10-20mumumoles of amino acid/mg. of protein; 40-150mumug. of carbon/mg. of protein was incorporated in 10min. from (14)C-labelled amino acid mixture. 3. Incorporation is stimulated by added ATP in the dark, but no added ATP is required when the system is illuminated. The cell-free plastid system is to some extent self-sufficient and does not normally require an added supernatant fraction or unlabelled amino acids. 4. Amino acid incorporation by plastids is inhibited by chloramphenicol, puromycin, actinomycin D, ribonuclease and deoxyribonuclease. It is suggested that the mechanism of protein synthesis in the cell-free plastids, and in the tissue generally, is basically the same as established for bacteria. Ribosomes and highspeed supernatant from this tissue were to some extent interchangeable with Escherichia coli ribosomes and supernatant in cell-free incubations. 5. Incorporation of amino acids by isolated plastids was stimulated by indol-3-ylacetic acid and kinetin, and, whereas incorporation normally proceeds for only 10-20min., the time-course was extended in the presence of these growth substances. It is suggested that hormones may be involved in the regulation of protein synthesis in plants.     

Protein synthesis in aging soybean cotyledons. Loss in translational capacity

Ribosomes and supernatant fractions from soybean cotyledons of different ages were prepared to study the Poly U (polyuridylic acid)-directed phenylalanine incorporation. Ribosomes from younger cotyledons were more effective in phenylalanine incorporation compared to ribosomes from older cotyledons. Similarly, the supernatant fractions from younger cotyledons were more efficient, resulting in enhanced incorporation, than the older cotyledons. Substitution of wheat embryo supernatant fraction for soybean cotyledon supernatant fraction resulted in a several fold increase in amino acid incorporation activity, in ribosomes from all ages of soybean cotyledons. Such increase in activity was particularly significant in the older cotyledons. From these experiments it is concluded that in aging soybean cotyledons there is a loss in translational capacity.     

Isolation and characterization of collagen messenger RNA*.

Chick embryo collagen-synthesizing polysomes were isolated by differential centrifugation. RNA extracted from these particles was chromatographed in oligo(dT)-cellulose solumns and the mRNA thus obtained characterized as collagen mRNA by its electrophoetical mobility in acrylamide gels (equivalent to 1.05 x 10-6 daltons) and its effect upon a cell-free system derived from Krebs ascites tumor cells. The incorporation of 3H-proline was markedly dependent upon rabbit reticulocyte initiation factors and inhibited by initiation inhibitors such as aurintricaboxilate and pyrocatechol violet. The incorporation product was characterized as collagen by its lack of tryptophan, digestibility by purified bacterial collagenase, and by its co-chromatography with unlabled chick collagen in Sephadex G-200 and CM-cellulose columns.     

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.     

Further purification of bovine spleen inhibitors of lymphocyte DNA synthesis (chalones).

Partially purified lymphocytic factors were obtained from boving spleen; these factors are non-cytotoxic and biologically active in vitro and in vivo: [3H]Thymidine incorporation into DNA of mitogen-stimulated mouse spleen cells in culture is inhibited; similar results are obtained with phytohaemagglutinin-stimulated peripheral human lymphocytes, where blast cells transformation is blocked. [3H]Thymidine incorporation into DNA of mitogen stimulated lymphocytes withdrawn from in vivo treated mice, is also reduced. The two factors in vitro and in vivo seem to act preferentially on mouse spleen cells compared to their action on liver, kidney and testicle cells in cluture, as far as thymidine incorporation into DNA is concerned. The following techniques were applied for their purification: 1. Homogenization of the fresh tissue in water, centrifugation, dialysis of the supernatant, centrifugation, fractionation of the supernatant by alcoholic precipitation and finally concentration in vacuo and lyophilization of the material soluble at 75% of alcohol yielded fraction F. 2. Preparation of an acetone powder from the spleen, extraction of the dry powder with water, then high speed centrifugation, followed by lyophilization of the supernatant produced fraction F'. Both fractions F and F' were further fractionated by chromatography on a Sephadex G75 column: 7 peaks were obtained (F1--F7). Biological activity was found in fraction F1, corresponding to high molecular weight material, and in fraction F6, corresponding to low molecular weight substances. By rechromatography on Sephadex G 75, it is easy to dissociate from F1 a small molecular weight fraction which might be similar to F6 as far as elution volume and biological properties are concerned.     

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.     

Effect of tannic acid on the synthesis of protein and nucleic acid by rat liver

1. As early as 1hr. after the intraperitoneal administration of tannic acid to rats, it could be demonstrated in the liver. At 3hr. the nuclear fraction contained the largest amount of tannic acid. 2. Nuclear RNA synthesis was inhibited in vivo 2hr. after the administration of tannic acid. Induction by cortisol of tryptophan pyrrolase was 90% inhibited at 24hr. 3. Incorporation of [1-(14)C]leucine into protein by liver slices from treated rats was decreased by 50% after 24hr. Its incorporation into postmitochondrial supernatant from treated animals was not inhibited. Incorporation into slices and postmitochondrial supernatants were inhibited in vitro by tannic acid. 4. The sequence of events: concentration of tannic acid in nuclei, inhibition of nuclear RNA synthesis, inhibition of protein synthesis and production of necrosis, is discussed.     

Amino acid incorporation in a cell-free system derived from rat liver studied with the aid of selenodiglutathione

Selenodiglutathione (GSSeSG), a potent inhibitor of elongation factor 2 (EF2) has been used to study amino acid incorporation in a rat liver cell-free system. While translocation of the ribosomes was inhibited by GSSeSG, ribosomes with a free acceptor site were still capable of incorporating one amino acid residue. From this the average number of amino acids incorporated per ribosomes was calculated to be 2--5. In this respect virtually no difference has been observed between ribosomes present on small or large aggregates. The time required for one translocation by all active ribosomes, and the time required for the incorporation of one amino acid (starting with aminoacyl-tRNA or amino acids) has also been determined. By incubation under conditions for amino acid incorporation, part of the ribosomes were completely inactivated whereas the rest remained as active as at the start of the incubation.     

In vitro Protein Synthesis by Polyribosomes of Peach Flower Buds at Different Physiological Stages

The in vitro phenylalanine incorporation by polyribosomes of peach flower buds (Prunus persica Stokes) during dormancy, dormancy break and flowering was investigated. Protein synthesis was measured using as catalyst either calf liver soluble factors or the ribosomal supernatant from the peach flower buds in the presence or the absence of the synthetic mRNA, polyuridylic acid. In the presence of polyuridylic acid, the activity of protein synthesis of dormant ribosomes is the same as that of ribosomes during dormancy break and flowering. The absence of synthetic messenger did not cause a change in activity. The ribosomal supernatant of dormant buds, but not of flowering buds, reduces the phenylalanine incorporation by polyribosomes from buds harvested at dormancy break.     

Studies on two groups of aminoacyl-tRNA synthetases from rat liver

Aminoacyl-tRNA synthetases from rat-liver cytoplasm were fractionated into two groups, characterized by their sedimentation coefficients of about 20S and 5S, respectively. These two groups of synthetases could be isolated from postmicrosomal supernatant either by gradient centrifugation, by gel filtration or by acid treatment at pH 5.2. Both groups were required for maximal amino acid incorporation in a cell-free system.