Role of aminoacyl-tRNA-synthetases in changes in the rate of protein biosynthesis in the rabbit liver during myocardial ischemia

Composition of high-molecular-weight aminoacyl-tRNA synthetases complexes from rabbit liver both in norm and after 12 h experimental myocardial ischemia (EMI) has been investigated. Partial redistribution of aminoacyl-tRNA synthetases activity from 1820 kD complex into 840 kD complex was observed in case of EMI which resulted in changes of protein biosynthesis rate in cell-free system.     

Murine mammary gland RNA directed synthesis of casein in a heterologous cell-free protein synthesis system.

A cell-free protein synthesis system derived from Ehrlich ascites tumor cell ribosomes (S30) plus rabbit reticulocyte tRNA was developed and the activity of the system was dependent on rabbit reticulocyte ribosomal salt (0.5 M KC1) wash factors, The exogenous mRNAs from BALB/c mouse liver and the mammary gland were translated with a high efficiency in this heterologous cell-free system. Furthermore, the RNA from the lactating mammary gland faithfully directed the synthesis of casein. The presence of mouse casein in the reaction product was identified by radioimmunoprecipitation with mouse casein antiserum, co-electrophoresis of the reaction product and mouse casein the urea-polyacrylamide gel and by electrophoresis in sodium dodecyl sulfate (SDS) polyacrylamide gel. The major portion of the lactating mammary gland RNA directed synthesis of the milk protein in the cell-free system appeared to be analogous to alphas casein,     

A factor converting monoribosomes into polyribosomes during protein synthesis in vitro

An extract was prepared from rabbit reticulocyte ribosomes after treatment with potassium chloride as described previously (Miller, Hamada, Yang, Cohen & Schweet, 1967). The participation of the extract in cell-free protein synthesis was studied. Purified polyribosomes were isolated and converted into monoribosomes by incubation in the cell-free protein-synthesis system. The monoribosomes were isolated and found to be unable to synthesize protein in the cell-free system. The addition of the ribosomal extract to the system stimulated protein synthesis. This was accompanied by the conversion of some of the monoribosomes into polyribosomes. The active component or components of the extract were shown to be protein.     

Distribution of aminoacyl-t-RNA-synthetase activity in rabbit liver cells during disruption of protein biosynthesis in experimental myocardia infarct

Distribution of the aminoacyl-tRNA synthetase activity has been studied in the normal rabbit liver cells and in the model of protein synthesis damage, i.e. under experimental myocardial infarction (EMI). The activity of a number of aminoacyl-tRNA synthetases in postmitochondrial and postribosomal extracts from rabbit liver homogenate has been determined to increase 12 h after EMI. Gel filtration of the postribosomal extract on Sepharose 6B shows that the activity of aminoacyl-tRNA synthetases is distributed among the fractions with Mr 1.82 x 10(6), 0.84 x 10(6) and 0.12 = 0.35 x 10(6). The first two fractions (high-molecular-weight aminoacyl-tRNA synthetase complexes) contain arginyl-, glutamyl-, isoleucyl-, leucyl-, lysyl- and valyl-tRNA synthetases, whereas the low-molecular-weight fraction contains alanyl-, arginyl-, glycyl-, phenylalanyl-, seryl-, threonyl-, tryptophanyl- and tyrosyl-tRNA synthetases. In a case of EMI all the aminoacyl-tRNA synthetases translocate from the complexes with Mr 1.82 x 10(6) into the complexes with Mr 0.84 x 10(6), what provided evidence for the possibility to regulate protein synthesis by changes in compartmentalization of aminoacyl-tRNA synthetases.     

Synthesis and transport of the precursor for the beta-subunit of rat liver F1-ATPase

The synthesis and intracellular transport of the beta-subunit of rat liver F1-ATPase was studied in a cell-free system, using free polysomal mRNA from rat liver and isolated rat hepatocytes. The beta-subunit of rat liver F1-ATPase is synthesized as a larger precursor form in rabbit reticulocyte lysate and then transported into isolated mitochondria in the absence of protein synthesis. In pulse experiments at 37 degrees C, the precursor of the beta-subunit reached a plateau 30 min after the pulse. The labeled mature beta-subunit appeared in the particulate fraction (containing mitochondria) after a time lag and increased almost linearly with time up to 40 min.     

Translational inhibition by heme of the synthesis of hepatic δ-aminolevulinate synthase in a cell-free system

Synthesis of δ-aminolevulinate synthase in a rabbit reticulocyte lysate system directed by total polysomes from the liver of allylisopropylacetamide-treated rats was studied with the combined use of [³H]leucine and a specific rabbit antibody. The protein synthesis observed in the cell-free system employed represented mainly the peptide chain elongation and its termination rather than the net synthesis involving initiation. Synthesis of δ-aminolevulinate synthase in this cell-free system was inhibited progressively with the increased addition of hemin; the synthesis was reduced to about 40% by about 30 μM hemin. Synthesis of total protein, however, was not significantly affected by the addition of hemin. The data obtained suggest that heme inhibits a peptide chain elongation step in the synthesis of δ-aminolevulinate synthase.     

Characterization of cell-free synthesis of collagen by lung polysomes in a heterologous system.

In normal lung growth, post-pneumonectomy lung growth, and in possibly several lung disorders, there are marked alterations in the density of collagen and changes in the rate of synthesis of collagen relative to the synthesis of other lung proteins. To provide a technology to begin to understand these changes at the molecular level, polysomes were prepared from rabbit lung and translated in a heterologous cell-free system including rabbit reticulocyte 0.5 M KCl ribosomal wash fraction and liver tRNA. Collagen was shown in the cell-free product by collagenase sensitivity, hydroxylation of incorporated proline by peptidyl prolyl hydroxylase, agarose gel chromatography, and sodium dodecyl sulfate acrylamide gel electrophoresis. The cell-free system was optimized with respect to K+, Mg2+, amino acids, and ribosomal wash fraction and used under conditions where total protein synthesis and collagen synthesis are linear with respect to time and amount of polysomes. Under these conditions, collagen synthesis was directed almost entirely by polysomes derived from the endoplasmic reticulum. Polysomes isolated from late fetal lung directed collagen synthesis at twice the rate (per polysome) as those polysomes isolated from adult lung. Similar changes were seen if lung tRNA replaced liver tRNA and if lung ribosomal wash fraction replaced reticulocyte wash fraction. Although these changes in cell-free lung collagen synthesis with tissue explants, further studies will have to be carried out to determine whether, in fact, age-related alterations in control of lung collagen synthesis are truly explained by these findings.     

Inactivation of eucaryotic ribosomes by the toxic plant proteins abrin and ricin

The effect of abrin and ricin on protein synthesizing systems from different sources was studied. The protein synthesis in a cell-free system from rabbit reticulocytes and from rat liver was strongly inhibited by the toxins, whereas a system from E. coli was not affected. Separate treatments of ribosomes and postribosomal supernatant from a rabbit reticulocyte lysate showed that the site of action of the toxins is on the ribosomes. The inactivation of the rabbit reticulocyte lysate by the toxins was a function of the incubation time and temperature. Protein synthesis was not necessary for the toxins to exert their effect. The data indicate that abrin and ricin act only on the eucaryotic type of ribosomes, and that they exert their effect by enzymatic action.     

Presence of translational inhibitory activity in partially purified extracts from two Petrocoptis species

The presence of translational inhibitory activity in partially purified extracts from several paleoendemisms from Spain was investigated. The precipitates from 40-80% (NH4)2SO4 fraction from Petrocoptis glaucifolia and Petrocoptis grandiflora displayed a strong inhibitory activity on the protein synthesis of cell-free extracts from rat liver, rabbit reticulocytes and yeast and to a much lower extent on the protein synthesis in isolated rat liver cells. The inhibitors seem to be proteins since they were precipitated by high salt concentrations, were non-dialysable and were inactivated by heat. Since the partially purified extracts did not show unspecific RNA-A or protease activities, the active compounds can be considered to belong to the plant ribosome-inactivating proteins.     

Alterations in albumin secretion and total protein synthesis in livers of thyroidectomized rats.

Perfused rat livers and isolated rat hepatocytes exhibited a 50% decrease in the secretion of both albumin and total secretory proteins after thyroidectomy. In contrast, synthesis of non-secretory proteins was decreased by only 20% from the rates observed in liver preparations from euthyroid rats. These observations suggested a disproportionate effect of thyroidectomy on the synthesis of secretory proteins compared with non-secretory proteins. Disproportionate decreases in the synthesis of albumin in other endocrine-deficient states such as hypophysectomy and diabetes had previously been shown to be associated with decreases of similar magnitude in the relative abundance of albumin-mRNA sequences. In contrast, thyroidectomy did not affect the activity or amount of albumin mRNA in total liver poly(A)-containing RNA when assayed by cell-free translation and by hybridization with complementary DNA, respectively. Furthermore, labelling experiments in vivo demonstrated that albumin synthesis represented 12.9 +/- 0.5% and 12.4 +/- 0.4% of total protein synthesis in livers of thyroidectomized and euthyroid rats respectively. Therefore the fall in secretion of albumin and total secretory protein after thyroidectomy did not appear to be a reflection of disproportionate decreases in the synthesis of these proteins. Instead, defects in steps involved in the post-synthetic processing and secretion of albumin are suggested. A number of comparisons, including ribosome half-transit times, the size distributions of total and albumin-synthesizing polyribosomes, and the fraction of RNA present as inactive ribosomes, provided evidence that the overall decrease in protein synthesis after thyroidectomy was not due to generalized alterations in translational processes. Instead, the decrease in total protein synthesis appeared to reflect the RNA content of the liver, which fell in proportion to th decrease in protein synthesis.     

Preparation of poly (A)-binding proteins from endoplasmic reticulum-containing subfractions of RAT liver cells and their use in mRNA purification

Summary Approximately 2% of the proteins solubilised from rat liver microsomes or rapidly sedimenting endoplasmic reticulum (RS-ER) adsorbed to poly(A)-Sepharose. The adsorption appeared to be selective for a few proteins, and proteins of different apparent molecular weights adsorbed from RS-ER and the microsomes. The proteins from RS-ER with affinity for poly(A) were coupled to Sepharose and used for the purification of mRNA from rabbit mammary glands. A portion of the RNA which did not adsorb to poly(U)-Sepharose adsorbed to protein-Sepharose and was active in a cell-free protein synthesis system.Deceased on May 30, 1976.     

Translation of chicken interferon messenger in a cell-free protein synthesis system and in a cell culture

A highly effective cell-free system for protein synthesis was obtained from rabbit reticulocytes and for the first time used for synthesis of biologically active chicken interferon. The optimal conditions for translation of its mRNA were developed. The translation efficacy in the cell-free system was 10-50 times higher than that in the culture of heterologous cells. The higher the purity level of RNA, the higher the translation level. With respect to poly (A+) RNA sedimenting in the sucrose gradient 9S the efficacy reached 2560 units per 1 microgram of RNA. By the content of poly (A), sequences and rate of the sedimentation, mRNA of the chicken interferon was similar to that of the human fibroblast cell interferon. The possible translation of mRNA of the chicken interferon at low concentrations of exogenic potassium ions in the cell-free system is explained by production of interferon in infected cells where the concentration of the intracellular potassium significantly decreases which is indicative of the mRNA interferon similarity with virus templates. It was found that only albino New Zealand rabbits, but also chinchilla may be used for preparation of the cell-free protein synthesizing system. Various exogenic templates in the mRNA-dependent cell-free system prepared from reticulocyte nonfractionated lysate by treatment with micrococcal nuclease stimulated the protein synthesis by 7-15 times.     

Structural alterations of peripheral nerve monogalactosylceramides during development and Wallerian degeneration

Reaction products of selenite with thiols were tested for an inhibitory effect on amino acid incorporation in a cell-free system derived from rat liver and on protein synthesis in intact P815 and L1210 cells. In the cell-free system maximum inhibition, up to 96%, was reached at about 10 microM selenium. In intact cells inhibitory effect varied depending on which reaction product or cell line was used. Maximum inhibition was obtained after 30 min of incubation with selenium concentrations ranging from 0.25 microM to over 7 microM. Selenite itself also inhibited protein synthesis of L1210 cells, but only after 90 min of incubation and starting at selenium concentrations of 2 microM. Inhibition of protein synthesis in intact cells was followed by cell death. Pre-incubation of the reaction products of a monothiol (2-propanethiol) and of a vicinal dithiol (2,3-dimercapto-1-propanol) in culture medium showed a rapid decrease of the inhibitory capability of the product from the monothiol, but not of the product from the dithiol. The results indicate that selenite and a thiol react to form products which have differential toxic effects to cells in vitro.     

Mapping the genes of the vaccinia virus, coding membrane proteins p34 and p40, by mRNA hybridization selection]

The HindIII--J HindIII-F fragments of the vaccinia virus DNA strain Lister have been analysed by the technique of mRNA hybridization selection with the subsequent translation in cell-free protein synthesizing system from the rabbit reticulocytes. The viral mRNA hybridizable with the HindIII--J fragment was shown to direct the synthesis of 30 kDa polypeptide in the cell-free system. This polypeptide was demonstrated to react specifically with antiserum to plasma membrane protein p34. The viral mRNA hybridizable with the HindIII-F fragment was shown to direct the synthesis of 37 kDa polypeptide in the cell-free system. This polypeptide reacts specifically with antiserum to major membrane protein p40.     

Inhibition of peptide chain initiation in lysates from ATP-depleted cells.I. Stages in the evolution of the lesion and its reversal by thiol compounds, cyclic AMP or purine derivatives and phosphorylated sugars.

Anaerobic incubation of rabbit reticulocytes at 37 degrees C in Krebs-Ringer solution supplemented with hemin but devoid of glucose resulted at the end of 1-2h in a drastic decline of their ATP content and an attendant arrest of protein synthesis. Subsequent provision of glucose and reoxygenation of the cells was followed by a rapid replenishment of the ATP pool, while resumption of protein synthesis was markedly delayed. This lag period could be considerably reduced by addition of 5-10 mM adenine or 2,6-diaminopurine to the incubation medium. Lysates prepared from ATP-depleted cells exhibited disaggregation of the polysomes and an inhibition of the nedogenously coded protein synthesis, when tested in a cell-free system supplied with an adequate ATP generator. Both alterations increased in severity with the progressive decay of the intracellular ATP pool. The early phase of partial inhibition following a 40-70% decrease of the cellular ATP level was fully reversible by fortifying the cell-free preparation with dithiothreitol or a suitable NADPH-generating system. Aternative, the inhibition could be also overcome by millimolar amounts of adenine, 2,6-diaminopurine and a variety of other purine derivatives or cyclic AMP. The effect of these compounds was unrelated to the endogenous cyclic AMP pool. Joint addition of both dithiothreitol and cyclic AMP or adenine was necessary for relieving the initiation block in lysates derived from cells depleted of 80-90% of their ATP content. On further aggravating the conditions of energy starvation, an additional requirement for phosphorylated sugars, e.g. glucose 6-phosphate or fructose 1,6-diphosphate, became apparent. ATP depletion brought about by exposing the cells to Antimycin A or 2,4-dinitrophenol resulted in a lesion which was indistinguishable from that induced by anaerobic incubation. On the other hand, energy deprivation in cell-free lysates from untreated reticulocytes, preincubated in the absence of an ATP-generating system failed to duplicate the deleterious effect of intracellular ATP depletion. Some aspects bearing on the biochemical mechanism of the lesion and its reversal are discussed in the light of the available data.     

In vitro translation and estradiol-17beta induction of Xenopus laevis vitellogenin messenger RNA.

Administration of estradiol-17beta to male Xenopus laevis induces synthesis and secretion by the liver of the egg yolk precursor protein vitellogenin. RNA extracted from livers of estradiol-17beta-treated Xenopus laevis directs the synthesis of the entire 200,000-dalton vitellogenin monomer in a cell-free protein synthesizing system derived from rabbit reticulocytes. Vitellogenin synthesized in vitro was isolated and quantitated by indirect immunoprecipitation and identified by comparison to authentic [14C]vitellogenin. The in vitro product and [14C]vitellogenin co-migrate on electrophoresis in sodium dodecyl sulfate-polyacrylamide gels and they exhibit identical immunoprecipitation curves. Xenopus laevis vitellogenin messenger RNA has a sedimentation coefficient of approximately 30 S in sucrose density gradients. It can be purified approximately 60-fold from cell RNA by poly(U)-Sepharose chromatography and therefore appears to contain a polyadenylate sequence. Vitellogenin mRNA and vitellogenin synthesis in vivo could not be detected in unstimulated male Xenopus laevis. The relative rate of vitellogenin synthesis and the level of vitellogenin mRNA were determined at various times following the administration of estradiol-17beta. Vitellogenin synthesis is maximal 12 days after estradiol-17beta administration when it comprises approximately 70% of cell protein synthesis. The level of vitellogenin mRNA and the intracellular rate of vitellogenin synthesis exhibit a close correspondence from 4 to 16 days after administration of estradiol-17beta.     

Characterization of an Initiating Cell-Free Protein Synthesis System Derived from Rabbit Brain

Abstract: Protein synthesis in the brain is known to be affected by a wide range of treatments. The detailed analysis of the mechanisms that are involved would be facilitated by the development of cell-free translation systems derived from brain tissue. To date, brain cell-free systems have not been fully characterized to demonstrate a capacity for initiation of translation. The following criteria were utilized to demonstrate that a cell-free protein synthesis system derived from rabbit brain was capable of initiation in vitro : (a) sensitivity of cell-free translation to the initiation inhibitor aurintricarboxylic acid (ATA); (b) binding of [³⁵S]Met-tRNA_f to 40S and 80S initiation complexes; (c) incorporation of labeled initiation methionine into high-molecular-weight proteins; and (d) the association of labeled exogenous mRNA with polysomes. The optimum conditions for amino acid incorporation in this system were 4 mM-Mg²⁺, 140 mM-K⁺, and pH 7.55. Incorporation was dependent on the addition of ATP, GTP, and an energy-generating system. Cell-free protein synthesis reflected the normal process, since a similar spectrum of proteins was synthesized in vitro and in vivo. This initiating cell-free translation system should have wide application in the analysis of the mechanisms whereby various treatments affect protein synthesis in the brain.     

Effect of Intravenous Administration of D-Lysergic Acid Diethylamide on Initiation of Protein Synthesis in a Cell-Free System Derived from Brain

An initiating cell-free protein synthesis system derived from brain was utilized to demonstrate that the intravenous injection of D-lysergic acid diethylamide (LSD) to rabbits resulted in a lesion at the initiation stage of brain protein synthesis. Three inhibitors of initiation, edeine, poly(I), and aurintricarboxylic acid were used to demonstrate a reduction in initiation-dependent amino acid incorporation in the brain cell-free system. One hour after LSD injection, there was also a measurable decrease in the formation of 40S and 80S initiation complexes in vitro, using either [35S]methionine or [35S]Met-tRNAf. Analysis of the methionine pool size after LSD administration indicated there was no change in methionine levels. Analysis of the formation of initiation complexes in the brain cell-free protein synthesis system prepared 6 h after LSD administration indicated that there was a return to control levels at this time. The effects of LSD on steps in the initiation process are thus reversible.     

Nonuniform size distribution of nascent peptides: The role of messenger RNA

The origin of the nonuniform size distribution of nascent rabbit globin peptides has been investigated in the reticulocyte lysate and wheat germ cell-free protein synthesis systems. Increasing the concentrations of the cellular components involved in protein synthesis failed to alter the elution pattern observed upon chromatographic analysis of reticulocyte lysate nascent chains. Nascent chains isolated from a globin messenger RNA-directed wheat germ cell-free system showed a nonuniform size distribution of nascent peptides similar to that of the rabbit reticulocyte nascent chains. These observations indicate that the nonuniformity of the globin nascent chains arises from a unique property of the messenger RNA being translated and not from limiting concentrations of a component or components of the reticulocyte protein synthesis system.