Addition Of Polyuridylic Acid Research Articles

Vicia sativaL. ‘Run-off’ and Purified Ribosomes: Polyphenylalanine Synthesis and Molecular Action of Ribosome-inactivating Proteins

A ‘run-off’ cell-free translation system (a 30000 × g supernatant; S 30) has been prepared from 4–5-d-old embryonic axes of Vicia sativa L., a plant lacking ribosome-inactivating protein activities which is very sensitive to certain RIPs but not to others. The system was able to generate a high rate of polyphenylalanine synthesis upon addition of polyuridylic acid. From this supernatant, purified ribosomes and a 100000 × g supernatant were prepared which were able to perform polyphenylalanine synthesis when mixed together (reconstituted system). The most important translation parameters were optimized in each case. Both the S 30 and the reconstituted system displayed differential sensitivities to certain RIPs. The purified RIP-inactivated ribosomes were able to release a 370 nucleotide rRNA fragment diagnostic for RIPs upon treatment of the isolated rRNA with acid aniline.

Protein synthesis by a cell-free preparation from the bird malaria, Plasmodium lophurae.

Cytoplasmic polyribosomes were isolated from the avian malaria parasite Plasmodium lophurae by lysis with 0.15% Triton X-100 followed by high speed centrifugation through a discontinuous sucrose gradient. Polyribosomes were protected from nuclease degradation using 100 mug/ml heparin or 50 mug/ml dextran sulfate. Cell-free incorporation of radioisotope-labeled amino acids required a pH 5 fraction (duck reticulocyte), Mg2+, and an energy-generating system. The protein synthesizing system was stimulated by the addition of polyuridylic acid. Optimum conditions for protein synthesis by the plasmodial system are described. The effects of drugs on the cell-free protein synthesizing system using duck reticulocyte and plasmodial ribosomes are reported.

Pituitary gland polyribosomal function in rats bearing pituitary hormone-secreting tumors

A cell-free system consisting of ribosomes and cytosol fraction, prepared from rat anterior pituitary tissue, was shown to be active in the incorporation of radioactive leucine into protein. The incorporation of radioactive amino acid resembled protein synthesis in its requirement for ribosomes, cytosol fraction, Mg 2+ and an energy source. The incorporation process was inhibited by puromycin. Sucrose density gradient centrifugation of the ribosome preparations revealed the presence of aggregates of ribosomes as well as single ribosomes. Polyribosomes isolated from pituitaries of control rats consistently incorporated more labeled amino acid into protein than did polyribosomes from pituitaries of rats bearing pituitary tumors. Incorporation by either population of polyribosomes was not changed when cytosol from the alternate source was used. Addition of polyuridylic acid caused an increase in the incorporation of [ 3H] phenylalanine by both polyribosome preparations, however, the functional difference between the two preparations was not eliminated. Sucrose density gradient analysis showed no difference in the sedimentation patterns of polyribosome preparations from pituitary glands of normal and tumor-bearing rats. It is concluded that pituitary polyribosomes from rats bearing hormone-secreting pituitary tumors are less efficient at protein synthesis in vitro than are polyribosomes from glands of normal rats. The defect is suggested to be at the ribosomal level, but the mechanism of this alteration in the ribosomal fraction has not been elucidated.

Inhibition of ribonucleic acid and of protein synthesis in the organs of rats and mice poisoned with α-amanitin

The synthesis of RNA in vivo, the activity of RNA polymerase of isolated nuclei, and the synthesis of protein in vivo have been studied in the liver and kidney of rats and mice, at various times after administration of α-amanitin. The synthesis of RNA is impaired reversibly in the liver of both species and irreversibly in mouse kidney, whereas it is almost unaffected in rat kidney. Almost parallel changes were observed in the activity of RNA polymerase II of isolated nuclei. Protein synthesis was decreased in mouse liver and kidney, and much less in rat organs. The administration of α-amanitin was followed by disaggregation of polysomes in mouse liver; microsomes isolated from the same organ had a reduced capacity of incorporating phenylalanine into protein in vitro, which could be restored by addition of polyuridylic acid.

Protein Synthesis in Cotyledons of Pisum sativum L

The changes in protein content of pea cotyledons have been followed during the period from 9 to 33 days after flowering. Initially protein content increased gradually with a rapid period of deposition occurring between days 21 and 27 after flowering. After the 28th day the rate of accumulation of protein declined as the seed dehydrated and matured. At maturity the pea cotyledon contained approximately 25% protein which was divided into albumins and globulins in the ratio of 1:1.4.Analytical data and the incorporation of exogenously supplied (14)C-leucine indicated that albumins were synthesized early in cotyledon development whereas globulin synthesis predominated with increasing maturity.Ribosomal preparations extracted from seeds during the period of rapid protein synthesis contained a high percentage of polysomes. Preparations from older cotyledons with a declining capacity for protein synthesis had few polysomes and an abundance of monosomes. The amino acid-incorporating capacity of ribosomal preparations from cotyledons of varying age was related to the polysomic content. The phenylalanine-incorporating capacity of ribosomal preparations from mature pea seed could be stimulated by the addition of polyuridylic acid. The distribution of polysomes and the in vitro incorporation data suggested that protein synthesis could be partially restricted by the availability of messenger RNA at maturity.However, reciprocal mixing experiments of supernatant and ribosomal fractions from cotyledons of different developmental age indicated that the supernatant fractions have varying capacities to stimulate in vitro amino acid incorporation. Thus the possibility of the regulation of protein synthesis at the translational level was not precluded.

Changes in polysomes of the developing rat brain.

Abstract— Rat brain polysomes were prepared from a deoxycholate‐treated postmito‐chondrial supernatant in the presence of 2% bentonite and 1 mg/ml of yeast RNA to prevent partial degradation during preparation. The polysomal preparations had an absorption maximum at 260 mμ and an absorption minimum at 235 mμ. The ratio of absorption maximum to minimum and the RNA to protein ratio were 1·58 and 1·06 respectively in 6‐day‐old rat brain polysomes. The sedimentation patterns showed six distinct peaks with sedimentation coefficients of 235S, 185S, 173S, 135S, 100S and 80S, indicating that these preparations have the characteristics of pure heavy polysomes. The rate of [14C]phenylalanine incorporation into brain polysomal protein was maximal at approximately 10 days of age and decreased thereafter. A similar progressive reduction with increasing age was found in the stimulation of phenylalanine incorporation by the addition of 60 μg/tube of polyuridylic acid. However, the incorporation of phenylalanine into young rat brain polysomes was usually greater even with the addition of polyuridylic acid than in the older animals. The comparative studies on sucrose density gradient centrifugation of polysomes between young and adult rat brains showed a considerable decrease of heavy polysomes in the older animals. The effect of various factors on the stability of brain polysomes from both ages has been studied. The rates of RNA, protein and acid‐soluble phosphorus release from polysomes of the adult rat brains were usually greater in the presence of high salt concentration, ethylenediaminetetra‐acetic acid and urea than those from the corresponding preparations of younger animals. On the basis of evidence obtained from the above results it suggested that the adult brain polysomes were more unstable than those of younger animals. The amount of polysomal RNA linearly increased up to the first 20 days after birth and then levelled off. The ratio of G + C/A + U of polysomal RNA was less in the young rat brains, falling to 1·30 as compared to 1·50 in older animals. The differences were statistically significant at less than a 1% level of confidence. Polysomal preparations also contained RNase, phosphomonoesterase, phospho‐diesterase and 5′‐nucleotidase activities which cannot be washed off. The specific activities of these enzymes were generally higher in young rat brains than those in the adult.

Lability of the protein biosynthesis system of the mouse kidney and its relationship to the regulatory effect of androgens.

ABSTRACT The amino acid (leucine, phenylalanine) incorporation system of the postmitochondrial fraction (S2) from the kidney of normal, castrated and testosterone propionate treated mice lost all of its activity on storage over-night at 5°C. The loss in activity was evident within one hour of storage at 4°C. Most of the amino acid incorporating activity was lost within 10 min on preincubation at 37°C. Addition of polyuridylic acid was not able to restore the activity. Neither castration nor the administration of androgen influenced the rate of loss of the amino acid incorporating activity. Separation of the soluble (S3) and microsomal (Ms) fractions and preincubation of either of the fractions produced loss in activity. The addition of poly U restored the activity of the preincubated Ms but not the preincubated S3 fraction. Storage of S2 at −20°C only retarded the rate of loss in activity. The addition of poly U to the enzyme system restored all of the phenylalanine incorporation activity for the castrated mice and most but not all of that of the androgen treated mice. The loss in activity was not accompanied by any detectable change in the RNA.

Protein Synthesis in a Cell-free System Derived from the Rat Parotid Gland

Abstract A cell-free amino acid-incorporating system has been prepared from rat parotid salivary glands and has been shown to possess activity comparable to that of a similarly constituted rat liver cell-free system. Administration in vivo of isoproterenol, prior to removal of the glands, reduced the secretory ribonuclease content, along with the other exportable proteins, to sufficiently low levels to permit the preparation of intact ribonucleoprotein particle and pH 5 enzyme fractions. The pH 5 enzyme fraction was tested separately, and shown to be as active as a corresponding rat liver fraction in the formation of amino acyl transfer RNA. Ultracentrifugal analyses of the ribonucleoprotein particle fraction, derived from rat parotid tissue, revealed a number of ribonuclease-sensitive peaks with sedimentation coefficients consistent with polyribosomal species. The optimal conditions for amino acid incorporation were established revealing an optimal Mg++ ion concentration of 10 to 14 mm as contrasted with a lower optimum for the liver system, and a less prolonged time course of incorporation than in the rat liver system. The addition of polyuridylic acid to the rat parotid system stimulated the incorporation of phenylalanine into protein by approximately 5-fold with no shift in the optimal Mg++ ion concentration. This was in contrast to the 2.5 fold stimulation, requiring an upward shift in the Mg++ ion concentration, seen in the rat liver system. These findings represent the first report of a cell-free protein synthesis system derived from a mammalian salivary gland, and also demonstrate a new technique for reducing damage to subcellular fractions in exocrine glands by their endogenous secretory enzymes.

Temperature-sensitive yeast mutant defective in ribonucleic acid production.

A single, recessive mutation in a nuclear gene confers a temperature-sensitive growth response in a mutant of Saccharomyces cerevisiae, ts(-) 136. The mutant grows normally at 23 C, but exhibits a rapid and preferential inhibition of ribonucleic acid (RNA) accumulation after a shift to 36 C, demonstrating a defect in stable RNA production. Cultures of the mutant which were shifted from 23 to 36 C display the following phenomena which indicate that messenger RNA (mRNA), as well as stable RNA production, is defective. The entrance of pulse-labeled RNA into cytoplasmic polyribosomes is even more strongly inhibited than is net RNA accumulation. The rate of protein synthesis, at first unaffected, decreases slowly; this decrease is paralleled by the decay of polyribosomes to monoribosomes with a half-time of 23 min. The polyribosomes which remain after a 30-min preincubation of the mutant at 36 C are active in polypeptide synthesis in vivo, whereas the monoribosomes which accumulate are not. Furthermore, ribosomes isolated from a culture of the mutant preincubated for 1 hr at 36 C are inactive in polypeptide synthesis in vitro, but can be restored to full activity by the addition of polyuridylic acid as mRNA. We conclude that mutant ts(-) 136 is defective either in the synthesis of all types of cytoplasmic RNA, or in the transport of newly synthesized RNA from the nucleus to the cytoplasm, and that the mRNA of a eucaryotic organism (yeast) is metabolically unstable, having a half-life of approximately 23 min at 36 C.

Étude de l'incorporation de la phénylalanine dans les protéines par un système acellulaire de foie de poulet en fonction de l'âge

Study of phenylalanine incorporation into proteins in a cell free system from chicken liver as a function of age The study of labeled phenylalanine incorporation into proteins synthetized in a system containing microsomes from liver of chicken embryos shows a high activity in the presence of endogenous messenger RNA and, after inactivation of this messenger RNA through preincubation, a strong stimulation upon addition of polyuridylic acid. On the contrary, the same study, performed in a system of microsomes from liver of adult chicken, shows only a slight activity in the presence of endogenous messenger RNA, and after preincubation, only a minute stimulation upon addition of polyuridylic acid. These results can be improved when the supernatant from embryos' liver is used as an enzyme source, rather than the supernatant from adult's liver. A study of the activity as a function of the age of the animals shows a sharp decrease between the liver of 2-week-old embryos and the liver of 1-day-old chicken, and then a progressive decrease down to the minimal values obtained with adult chicken liver (either for direct incubation, or after preincubation, in the presence or absence of polyuridylic acid). Ribosomes, prepared from microsomes through deoxycholate treatment, show an endogenous activity whose absolute value is superior to that observed with the corresponding microsomes, except in the case of embryos where it is equal. Similarly, the ratio of stimulation upon addition of polyuridylic acid is always higher with the ribosomes. After inactivation of endogenous messenger RNA through preincubation, stimulation upon addition of polyuridylic acid remains constant and equal to that observed with the ribosomes of embryos' liver, independent of the age of the animals.

GTP and Protein Synthesis in Cell-Free Systems

Abstract Two supernatant fractions, T1 and T2, have been isolated and partially purified from rat liver and rabbit reticulocytes. In a cell-free system programmed with polyuridylic acid, both fractions are needed for phenylalanine polymerization. T1 factor and GTP are required for the enzymatic binding of phenylalanil-tRNA to template charged ribosomes. In the course of the binding reaction, GTP is hydrolyzed while no dipeptide formation can be detected. T2 factor coincides with a ribosome-linked GTPase, which is not stimulated by the addition of polyuridylic acid and phenylalanil-tRNA.

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 x 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.

Studies on the Role of Ribosomes in the Regulation of Protein Synthesis in Hypophysectomized and Thyroidectomized Rats

Abstract Polyribosomes were obtained from the livers of hypophysectomized and control rats. Although sucrose gradient analysis disclosed that polyribosome profiles of the livers of hypophysectomized animals were identical with those of livers derived from intact controls, the rate of amino acid incorporation into hepatic protein in vitro was lower in the polyribosomes derived from hypophysectomized animals. Furthermore, the stimulation of phenylalanine-14C incorporation that followed the addition of polyuridylic acid was less when the disaggregated polyribosomes were obtained from hypophysectomized rats than when similar preparations obtained from control animals were used. Similar findings were obtained with polyribosomes from thyroidectomized rats. These studies suggest that hypophysectomy and thyroidectomy influence protein synthesis predominantly by action at the level of the ribosome.

Amino Acid Incorporation and Aminoacyl Transfer in a Wheat Embryo System

Abstract An amino acid-incorporating system has been obtained from ungerminated wheat embryos. This system has very low endogenous activity, but phenylalanine incorporation can be stimulated by the addition of polyuridylic acid. The poly U-stimulated system is similar to those from other sources in that it requires ribosomal particles, supernatant fluid, soluble ribonucleic acid, adenosine triphosphate, guanosine triphosphate, a nucleoside triphosphate-regenerating system, and magnesium and potassium ions for maximal activity. Puromycin and ribonuclease cause considerable inhibition of phenylalanine incorporation while chloramphenicol has little effect. The polymerization of phenylalanine from phenylalanyl-soluble ribonucleic acid in the presence of polyuridylic acid requires ribosomes, Mg++, K+, GTP, its generating system, and a factor present in the supernatant fraction. This aminoacyl transfer factor from wheat embryos has been partially purified by ammonium sulfate precipitation and DEAE-cellulose column chromatography. The specificity of the ribosomes for the supernatant factor in the transfer reaction was studied with ribosomes from Escherichia coli, guinea pig liver, wheat embryo, and tobacco leaf chloroplasts. The mammalian and wheat ribosomes could interchange their supernatant factors to give aminoacyl transfer but were not active with the E. coli factor. E. coli ribosomes could use only the homologous supernatant fraction. The tobacco leaf chloroplast ribosomes were tested with the wheat supernatant fluid and were found to be active.

The effect of chick-liver ribonucleic acid on 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.

INHIBITION OF PHENYLALANINE INCORPORATION BY POLYURIDYLIC ACID.

ADDITION of polyuridylic acid (poly U) to cell-free systems prepared from a number of tissues results in enhanced incorporation of phenylalanine into polypeptide material1–4. Arnstein et al.2 noted that, when poly U was added to a reticulocyte ribosome system, the incorporation of other amino-acids was not inhibited and they concluded that natural messenger RNA (m-RNA) was not displaced from the ribosomes by the poly U. They questioned whether the poly U effect on phenylalanine incorporation had any physiological significance. Weinstein et al.5 later showed that incorporation of some amino-acids was inhibited when poly U was added to reticulocyte ribosomes. The present report concerns an instance when poly U, added to rat liver polysomes prepared in the absence of magnesium, actually inhibited phenylalanine incorporation. It is argued that this anomalous occurrence, paradoxically, strengthens the concept that poly U stimulation of phenylalanine incorporation is a reflexion of physiological events.

Function of aggregated reticulocyte ribosomes in protein synthesis

The ribosome fraction of rabbit reticulocytes contains ribosomal units with a sedimentation coefficient of 80 s and some smaller components. In addition there is a spectrum of at least five more rapidly sedimenting fractions, comprising of the order of 30% or more of the material. The fast fractions are considered to be aggregates of ribosomes consisting of up to six ribosomal units. They disintegrate into the 80 s ribosomal units upon incubation with RNase. The fast fractions, separated from the slow fraction by sucrose gradient centrifugation, are shown to be more active both in whole cell and in cell-free incorporation of radioactive amino acids. Incorporation of [14C]phenylalanine is stimulated by the addition of polyuridylic acid as messenger RNA. The stimulation of activity is maximum for unaggregated ribosomes. Part of the active products of stimulation appear to be aggregates. The results indicate that many of the aggregated ribosomes contain messenger RNA, but are not receptive to added messenger RNA; whereas many of the unaggregated ribosomes do not contain messenger RNA, but are receptive to added messenger RNA molecules. The simplest interpretation is that the linkage of the ribosomes into aggregates is due to the messenger RNA itself. The findings support a theory of protein synthesis whereby several ribosomes can operate simultaneously on one molecule of messenger RNA.