Amino Acid-incorporating Activity Research Articles

The use of protein synthesis inhibitors in the estimation of the contribution of halophilic archaebacteria to bacterial activity in hypersaline environments

Antibiotics affecting protein synthesis were used to differentiate between the activity of different groups of organisms (halophilic archaebacteria, eubacteria and eukaryotes) in water samples from hypersaline ecosystems. Anisomycin (inhibiting both archaebacterial halophiles and eukaryotes) can be used to quantitate the contribution of the archaebacterial halophiles to amino acid incorporation by the microbial community, when cycloheximide (inhibiting eukaryotic protein synthesis, but not affecting halobacteria) is used as a control. Both in saltern ponds at salinities above 300 g/1 and in Dead Sea surface water more than 95% of the amino acid incorporation activity was abolished by anisomycin, but not by cycloheximide. Inhibition by anisomycin was well correlated with inhibition by low concentrations of bile salts, which specifically affect bacteria of the Halobacterium group. Chloramphenicol (an inhibitor of eubacterial protein synthesis) quantitatively inhibited amino acid incorporation in saltern brines of relatively low salinity, but also caused significant (28–42%) inhibition at high salinities. Erythromycin was also found valuable in the estimation of activities of the different bacterial groups.

The use of protein synthesis inhibitors in the estimation of the contribution of halophilic archaebacteria to bacterial activity in hypersaline environments

Antibiotics affecting protein synthesis were used to differentiate between the activity of different groups of organisms (halophilic archaebacteria, eubacteria and eukaryotes) in water samples from hypersaline ecosystems. Anisomycin (inhibiting both archaebacterial halophiles and eukaryotes) can be used to quantitate the contribution of the archaebacterial halophiles to amino acid incorporation by the microbial community, when cycloheximide (inhibiting eukaryotic protein synthesis, but not affecting halobacteria) is used as a control. Both in saltern ponds at salinities above 300 g/1 and in Dead Sea surface water more than 95% of the amino acid incorporation activity was abolished by anisomycin, but not by cycloheximide. Inhibition by anisomycin was well correlated with inhibition by low concentrations of bile salts, which specifically affect bacteria of the Halobacterium group. Chloramphenicol (an inhibitor of eubacterial protein synthesis) quantitatively inhibited amino acid incorporation in saltern brines of relatively low salinity, but also caused significant (28–42%) inhibition at high salinities. Erythromycin was also found valuable in the estimation of activities of the different bacterial groups.

Estimation of protein synthesis in fish larvae using an in vitro polyribosome assay

An in vitro method for use in studies of protein synthesis in fish larvae is described. Cod larvae were used. It is shown that measurement of amino acid incorporation activity is possible with ribosomes from pooled samples of 50–100 larvae. In assays of ribosomes from whole cod larvae with a high growth rate, the capacity for incorporating amino acids into protein was shown to be about 20 times higher than in ribosomes isolated from cod muscle.

Effects of insulin on the fine structure of hepatocytes from winter-acclimatized carps: studies on protein synthesis.

Male and female winter-acclimatized carps were injected with insulin. This treatment resulted in a sharp decrease in the liver glycogen content. Although an increase in the ribosomal RNA level was also observed, a cell-free system obtained from the hormone-treated fish exhibited less amino acid incorporation activity as compared to the control fish. However, polysomes from insulin-treated fish exhibited a higher amino acid incorporating activity when a soluble fraction of untreated winter carps was used. Insulin induced a profound change in the cytoarchitecture of the winter carp hepatocyte. The cytoplasm and nuclei showed all the features of the summer carp liver cell. The nucleolar components were totally intermingled suggesting a high rate of gene expression as in the case of the summer-acclimatized fish.

Structural integrity of DNA and translational integrity of ribosomes in nuclease-treated cell-free protein synthesizing systems prepared from wheat germ and rabbit reticulocytes.

After treatment at a microsomal nuclease concentration too low to reduce the endogenous amino acid-incorporating activity of freshly prepared reticulocyte lysate, there is little, if any, intact 26 S RNA left in the ribosomes of either wheat germ or rabbit reticulocyte cell-free protein synthesizing extracts. The primary scissions, probably at highly exposed sites in the rRNA of plant and animal ribosomes, produce two fragments which remain complexed until thermal denaturation reveals "hidden breaks." Molecular weights of the fragments are approximately 0.5 x 10(6) and 0.8 x 10(6) in the case of wheat, and 0.4 x 10(6) and 1.3 x 10(6) in the case of rabbit. There is little perceptible degradation of 5 S, 5.8 S, and 18 S rRNA, or of tRNA in the same extracts. Even though limited degradation of 26 S rRNA by a reticulocyte nuclease has been reported to severely impair the translational mechanism in reticulocyte ribosomes, micrococcal nuclease-induced degradation of rRNA, whether limited or extensive, does not seriously impair the ability of reticulocyte lysates to discriminate, by selective polypeptide synthesis, between complex populations of cellular mRNA. In an allied study, it is shown that under conditions well suited to recovery of the 5.8 S/26 S rRNA complex, with its naturally occurring hidden break, 5 S/18 S rRNA complexing is not detectable in the RNA or metabolizing embryos, nor in the RNA from untreated or nuclease-treated protein synthesizing extracts from wheat and rabbit. The significance of this finding is briefly elaborated in relation to the suggestion that 5 S rRNA may interact with the M2(6)A-m2(6)A hairpin near the 3'-end of 18 S rRNA.

Quantities and activities of ribosomes in posterior silk glands of the normal and Nd silkworms.

Two ribosomal fractions were prepared from the posterior silk glands of the norml and Nd mutant silkworms. The amounts of two ribosomal fractions of the Nd silkworm were reduced to 4.4 and 5.8%, respectively, of the normal one. The amino acid incorporation activity of the Nd cell-free system containing two ribosomal fractions corresponded to 15 and 13%, respectively, of the normal cell-free system containing the identical fraction. The reduced activity of the Nd cell-free system was mainly ascribable to ribosomes including endogenous mRNA.

Soluble factors and increase of protein synthesis in regenerating liver

1. 1. Synthesis of leucyl- and phenylalanyl-tRNA increased under various experimental conditions in cell sap obtained from regenerating rat liver. 2. 2. Activities as well as affinities for the substrates and ATP requirements of leucyl-tRNA synthetase were essentially the same in normal and regenerating livers. 3. 3. The difference between normal and regenerating cell sap disappeared on addition of a large excess of normal tRNAs. 4. 4. The increased amino acid incorporation activity by cell sap from regenerating livers does not seem to depend on concurrent increased activity of amino acyl synthetase, but might be associated with changes in tRNA.

Effect of polyamines on cell-free protein synthesizing systems from rat cerebral cortex, cerebellum and liver

The naturally occurring polyamines spermine and spermidine stimulate in low concentrations the amino acid-incorporating activity of cell-free systems from cerebral cortex, cerebellum and liver from the rat to a significant degree, but in high concentrations they have strong inhibiting effects on these systems. Maximal polyamine stimulation is observed with suboptimal Mg 2+ concentrations in the reaction mixtures; increasing Mg 2+ to 15 mM results in an inhibition of cell-free protein synthesis by polyamines even at low concentrations. The stimulating effect of spermine and spermidine involves a facilitated binding of template RNA and aminoacylated tRNA to the ribosomes. In contrast, the aminocylation of tRNA is not influenced. These data indicate that spermine as well as spermidine have regulatory functions during the translational process in cerebral cortex, cerebellum and liver. They seem to interact with ribosomal functions and to cause a shift in the optimal Mg 2+ concentration required for the synthesis of brain and liver proteins.

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.

An efficient mRNA-dependent translation system from reticulocyte lysates.

A simple method is described for converting a standard rabbit reticulocyte cell-free extract (lysate) into an mRNA-dependent protein synthesis system. The lysate is preincubated with CaCl2 and micrococcal nuclease, and then excess ethyleneglycol-bis(2-aminoethylether)-N,N'-tetraacetic acid is added to chelate the Ca2+ and inactivate the nuclease. Lysates treated in this way have neglibible endogenous amino acid incorporation activity, but 75% of the activity of the original lysate can be recovered by the addition of globin mRNA. The efficiency of utilisation of added mRNA and the sensitivity of the system are both very high. No residual nuclease activity could be detected, and the tRNA is functionally unimpaired. Several different species of mRNA have been shown to be translated efficiently into full-sized products of the expected molecular weight up to about 200000, and there is no detectable accumulation of incomplete protein products. The efficient translation of RNA from two plant viruses (tobacco mosaic virus and cowpea mosaic virus) required heterologous tRNA.

Studies on the amino acid-incorporating activity of native rat liver rough membrane and that reconstituted in vitro.

The amino acid-incorporating activities of free polyribosomes, rough membranes and rough membranes reconstituted in vitro, derived from rat liver, were compared. The amino acid-incorporating activity of the two membrane fractions were very similar in their response towards changes in pH, Mg2+ concentration and temperature, but differed from the response of the amino acid-incorporating activity of free polyribosomes. Free polyribosomes irreversibly lost part of their amino acid-incorporating capacity after they had become bound to rough membrane, from which the original ribosomes had been removed. Ribonuclease activity present in the membrane fraction may be responsible for this loss.

Formation, Size, and Solubility in Chloroform/Methanol of Products of Protein Synthesis in Isolated Mitochondria of Rat Liver and Zajdela Hepatoma

The number, size, solubility in chloroform/methanol and some aspects of the formation of the components labeled by radioactive amino acids in isolated mitochondria of rat liver and Zajdela hepatoma were studied. Isolated mitochondria were labeled with radioactive amino acids under various conditions, and the distribution of radioactivity in sodium dodecylsulfate-polyacrylamide gels after electrophoresis of mitochondrial membrane fraction was analysed. 1. Isolated mitochondria of rat liver and Zajdela hepatoma incroporated radioactive amino acids almost exclusively into the membrane fraction. Electrophoretic analysis of this fraction revealed the presence of 15 distinct peaks of radioactivity with corresponding apparent molecular weights of 10 000 to 58 000. The electrophoretic mobility of the labeled components was identical and the general pattern of the radioactivity distribution in the gel for the rat liver and the tumour mitochondria was very similar. 2. Components of the membrane fraction of rat liver mitochondria labeled in vitro displayed an unequal solubility in acidic (2 mM HC1) chloroform/methanol (2/1) mixture; as detected by sodium dodecylsulfate-polyacrylamide gel electrophoresis a single labeled component with apparent molecular weight of 10 000 was soluble in neutral chloroform/methanol. 3. Inverse relation was observed between amino acid incorporation activity of isolated mitochondria and the portion of the label incorporated into the component with apparent molecular weight 10 000. The identity of this component with that soluble in neutral chloroform/methanol mixture has been indicated. 4. The rate of incorporation of [3H]leucine by isolated Zajdela hepatoma mitochondria into the components with lower (10 000-25 000) apparent molecular weights decreased with time, whereas that into components with higher (above 25 000) apparent molecular weight remained approximately constant within the time interval tested (30 min). 5. From the total radioactivity incorporated into the membrane fraction during 5-min pulse labeling of isolated Zajdela hepatoma mitochondria by [3H]leucine up to 25% was recovered in the region of the gel corresponding to a component with apparent molecular weight 10 000. After 25 min chase the radioactivity in this region decreased about 3.5 times while the specific radioactivity of the total membrane fraction did not change significantly. The pattern of radioactivity distribution observed after the pulse was preserved by chloramphenicol. 6. Unlabeled sonicated mitochondria or postribosomal supernatant from rat liver regenerating in the presence of chloramphenicol were incubated with neutral chloroform/methanol extract of in vitro with [14C]leucine labeled rat liver mitochondria. After this incubation several labeled components with apparent molecular weights above 10 000 were recovered in the electrophoreograms of the originally unlabeled fractions.

The effect of some antibiotics on protein synthesis activity of in vitro reconstituted rough membrane from rat liver

In order to learn about the ribosome-membrane interaction, rough membranes have been stripped from their ribo- somes by different methods and experiments have been carried out to reconstitute the rough membrane from these stripped membranes and polyribosomes [2,3]. Ribosomes do reattach to the stripped rough membrane but the major question is whether the reconstituted rough membrane is identical to the original rough membrane before it was stripped. There are several reports about some differences between the amino acid incorporation activity of free polyribosomes compared to that of the rough membrane ribosomes, differences such as optimal Mg” concentration [4] and sensitivity towards pro- tein synthesis inhibitors [S] . In this communication we compare the effect of some inhibitors on protein synthesis promoted by rough to that of in vitro reconstituted rough membrane. 2. Materials and methods Preparation of rat liver fractions: Free polyribo- homes (FP), rough (RM) and smooth (SM) endoplas- mic membranes were prepared as described by Czosnek and Hochberg [6]. For reconstitution purpose, rough membrane was stripped using the KCl-puromycin method (RMst) and free polyribosomes were bound to this stripped rough membrane according Borgese et al. (RMrec) [3]. The different membrane fractions were pelleted and suspended in 5 ml 2 M sucrose TKM (Tris-HCl pH=7.6,50 mM; KCl. 25 mM;

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.

Cytoplasmic Type 80 S Ribosomes Associated with Yeast Mitochondria: II. EVIDENCE FOR THE ASSOCIATION OF CYTOPLASMIC RIBOSOMES WITH THE OUTER MITOCHONDRIAL MEMBRANE IN SITU

Abstract Electron microscopic examination of growing spheroplasts prepared from Saccharomyces cerevisiae revealed ribosome-like particles of 150 A diameter aligned along the outer mitochondrial membrane. Ribosome-like particles were also seen associated with membranes of the endoplasmic reticulum and the outer nuclear membrane. Vacuolar and plasma membranes, however, did not appear to have attached particles. When incubated in an amino acid-incorporating system containing pH 5 fraction, mitochondria isolated in the presence of 10 mm Mg2+ were capable of incorporating [3H]leucine into trichloroacetic acid-insoluble material. About 80 % of the incorporation was inhibited by cycloheximide, an inhibitor of cytoplasmic protein synthesis, indicating that the majority of the amino acid-incorporating activity was due to cytoplasmic ribosomes associated with mitochondria. Mitochondria-bound cytoplasmic ribosomes were capable of vectorial release of nascent chains as shown by the distribution of puromycin-discharged polypeptides: half remain associated with the mitochondria and were releaseable by detergent, and half were released directly into the medium. These results are comparable to data reported for the distribution of puromycin-released nascent polypeptide chains with preparations of rough endoplasmic reticulum from rat liver. About one-third of the mitochondria-associated cytoplasmic ribosomes could be released by incubating mitochondria in a buffer of high ionic strength. The remaining two-thirds, however, were released only after reacting their nascent polypeptide chains with puromycin. This result suggests a role for the nascent polypeptide chains in the attachment of cytoplasmic polysomes to mitochondria. Cytoplasmic polysomes can be released intact from isolated mitochondria by extraction with Triton X-100.

Dietary amino acids: effect of depletion and recovery on protein synthesis in vitro in rat skeletal muscle and liver.

1. Rats were given diets containing 200 g/kg of a complete or incomplete amino acid mixture or of high- or low-quality proteins. After 6 d the amino acid-incorporating activity of ribosomes from skeletal muscle and liver was studied.2. The level of isotope incorporation relative to ribosomal RNA was similar for casein supplemented with methionine and for a complete amino acid mixture with the composition of whole-egg protein. Per wet weight of tissue there was a significant decrease after feeding with the complete amino acid mixture.3. There was a significant decrease in activity after feeding with amino acid mixtures deficient in lysine, methionine or tryptophan. In skeletal muscle, but not in liver, the ribosomal activity was less than that obtained with wheat gluten. Activity per wet weight of both tissues was less than that obtained with wheat gluten.4. Refeeding with methionine for 1 d resulted in complete restoration of ribosomal activity and activity per wet weight in skeletal muscle.5. After lysine deficiency, protein synthesis per unit wet weight of both tissues and ribosomal activity in liver were not restored after 2 d of refeeding. Recovery of ribosomal activity in skeletal muscle was complete after 1 d.6. Rats receiving the 200 g casein/kg diet supplemented with methionine at daily energy levels of 263, 176, 141 and 106 KJ (62.6, 42.1, 33.7 and 25.3 kcal) showed no changes in ribosomal activity, but there was a significant decrease in activity per wet weight when 106 KJ were given.

Protein synthesis by perfused hearts from normal and insulin-deficient rats. Effect of insulin in the presence of glucose and after depletion of glucose, glucose 6-phosphate and glycogen.

In the absence of glucose, insulin stimulated the incorporation of (14)C-labelled amino acids into protein by perfused rat hearts that had been previously substantially depleted of endogenous glucose, glucose 6-phosphate and glycogen by substrate-free perfusion. This stimulation was also demonstrated in hearts perfused with buffer containing 2-deoxy-d-glucose, an inhibitor of glucose utilization. It is concluded that insulin exerts an effect on protein synthesis independent of its action on glucose metabolism. Streptozotocin-induced diabetes was found to have no effect either on (14)C-labelled amino acid incorporation by the perfused heart or on the polyribosome profile and amino acid-incorporating activity of polyribosomes prepared from the non-perfused hearts of these insulin-deficient rats, which show marked abnormalities in glucose metabolism. Protein synthesis was not diminished in the perfused hearts from rats treated with anti-insulin antiserum. The significance of these findings is discussed in relation to the reported effects of insulin deficiency on protein synthesis in skeletal muscle.

Biogenesis of mammalian mitochondria in the renoprival kidney: I. Amino acid incorporation and respiratory activity

Changes in the yield of mitochondrial protein, in the incorporation of leucine into mitochondrial proteins, and in the respiratory activity of isolated mitochondria were determined in the remaining kidney (renoprival kidney) of the rat during the first 72 hr postmononephrectomy. At 24, 48, and 72 hr the yield of mitochondrial protein isolated from the renoprival kidney increased 13, 23, and 34%, respectively, whereas renal mass increased 9, 14, and 19%. Incorporation of [ 3H]-leucine in vivo into total mitochondrial protein was increased 96 and 130% over control at 12 and 24 hr, respectively. Incorporation of leucine in vitro by mitochondria was increased 27% over control at 24 hr; chloroamphenicol, but not cycloheximide, inhibited the in vitro incorporation. At 24 hr, the rate of O 2 consumption (state 3) was decreased, but P O ratios, respiratory control values, and glutamic and succinic dehydrogenase activities were unaltered. The content of pyridine nucleotide, cytochromes a, a 3, and c was decreased at this time; these activities approached normal values by 72 hr. The results suggest that during the 24 and 48 hr postmononephrectomy there is an increase in mitochondrial protein and specific changes in mitochondrial function which may be indicative of a proliferation of mitochondria in the renoprival kidney. This system should be valuable in investigating the events involved in mitochondrial biogenesis in mammalian cells.

A cell-free system for protein synthesis from newly fertilized eggs of Drosophila melanogaster

Optimal conditions for cell-free protein synthesis using post-mitochondrial supernatant preparations from newly fertilized eggs of Drosophila melanogaster have been determined. The most active extracts were prepared using 75 mM KCl in potassium N-2- hydroxyethylpiperazine-N′-2- ethane sulfonate (pH 7.4). The optimal pH of the incubation medium was found to be 7.0. Dithiothreitol was more effective than other thiol reagents in enhancing amino acid incorporation by the system and was used in both the extraction and incubation media. A Mg 2+ concentration of 4 mM in the incubation system was found to represent a well-defined optimum. The monovalent cation requirement for optimal incorporating activity of the system is complex: NH 4 +, K + and Na + were all found to be required at a total concentration of 91 mM. Under the optimal conditions defined, the amino acid-incorporating activity of the system is comparable to the activities of mammalian and other insect systems. The Drosophila system is of interest since it apparently is active in cell-free initiation of polypeptide chains — as indicated by its sensitivity to low concentrations of NaF or aurintricarboxylic acid.

Biochemical studies on the mechanism of action of liver poisons I. Inhibition of protein synthesis

Administration of ethionine to rats was followed by a rapid decline in the uptake of labeled amino acids into the liver proteins. The magnitude of the decrease in the extent of the protein labeling remained unaltered when different radioactive amino acids were used as tracers, or when the amount of the amino acid injected was increased from a tracer dose up to a level in excess of the endogenous pool size. It was inferred that the reduced amino acid incorporation in vivo was not attributable to isotope dilution by a possible elevation of the free amino acid pools, but reflected an inhibition of protein synthesis. In rats treated with ethionine the derangement of protein synthesis in vivo exhibited an earlier onset and a faster time course than the decay of the cell-free, endogenously coded amino acid incorporation coinciding with an enhancement of the polyuridylic acid-directed phenylalanine polymerization and a disaggregation of the polysomes. This discrepancy between the inhibitory effects of ethionine observed in the intact animals and in the cell-free preparations was particularly prominent in male rats in which the dissociation of the polysomes and the deterioration of the cell-free protein-synthesizing capacity were much less pronounced than in the female rats. In contrast, the extent of hepatic ATP depletion was equally severe in both sexes. Injection of adenine or its nucleotide congeners together with ethionine into female rats prevented the fall of the liver ATP level as well as the decay of the protein-synthesizing capacity as measured in vitro, but failed to relieve appreciably the inhibition of protein synthesis in vivo. Paralell examination of the polysome patterns revealed that the exogenously supplied ATP precursors and ethionine induced a synergistic inhibition of protein synthesis in vivo, different from the injury caused by ethionine alone. This inhibition, characterized by the preservation of the intact polysomal structure, suggested an interference with the completion of the nascent peptide chains as the underlying mechanism of the phenomenon concerned. A similar type of inhibition was obtained by simultaneous administration of adenine mucleotides and an excess of methionine or other amino acids. The overall data are consistent with the interpretation that the synergistic block of peptide chain elongation “freezes” the polysomal assembly and thereby prevents the expression of the initiation lesion inherent in the deleterious effect of ethionine on the protein-synthesizing system. This explanation is borne out by the observation that pretreatment with a mixture consisting of 3′(2′)-AMP and either ethionine or methionine obviated the carbon tetrachloride-induced disaggregation of the polysomes and the concomitant decline of the cell-free amino acid-incorporating activity. The possible mechanisms underlying the noxiuos effects of ethionine on protein synthesis are discussed with special reference to the crucial role of ATP depletion in the pathogenesis of ethionine poisoning.