Physiology of rat-liver polysomes: Protein synthesis by stable polysomes

Certain qualitative aspects of protein synthesis in the livers of starved, starved-re-fed and actinomycin D-treated rats have been examined by polyacrylamide-gel electrophoresis. Animals were exposed to a mixture of (14)C-labelled acids for 18-20min. and killed, and an ultrasonic extract of newly formed protein in microsomal vesicles was prepared and examined by gel electrophoresis. In normal and starved-re-fed animals, 27% of the newly synthesized protein was albumin. During starvation, when RNA synthesis was decreased, the percentage of newly formed protein as albumin rose. After actinomycin D treatment of starved-re-fed rats, when only stable messenger RNA persisted in the cytoplasm, albumin synthesis increased to 63% of the total. This finding suggested that albumin was the primary protein synthesized on stable messenger RNA.     

Protein synthesis by membrane-bound and free ribosomes of the developing rat cerebral cortex

1. Rates of RNA and protein synthesis were measured in rat cerebral-cortex slices, and compared with amino acid incorporation into protein by membrane-bound and free ribosomes from the same tissue, in the first 3 weeks of life. 2. A rapid age-dependent decline in the incorporation of labelled precursors into both RNA and protein was observed, which was more marked for amino acid incorporation into protein. 3. Although membrane-bound ribosomes comprise only a small fraction of total ribosomes, they were more active in incorporating amino acids into protein than were free ribosomes, especially immediately after birth. The decline in activity with age was more marked in the membrane-bound fraction than in free ribosomes. This loss of activity was largely independent of alterations in soluble factors or endogenous mRNA content and appeared to involve some alteration of the function of the ribosome itself, with relatively small alterations in the ratio of membrane-bound to free ribosomes. 4. Thyroidectomy, performed soon after birth, had no effect on the incorporation of radioactive precursors into RNA or protein by either slices or the cell-free preparations during the first 3-4 weeks of life.     

Protein synthesis of the sponge Geodia cydonium: characterization of the system

The ribosomal population of the sponge Geodia cydonium has been examined. The monosomes have a sedimentation constant of 80 S, the sizes of the subunits are approximately 60 S and 45 S respectively. The polyribosomes contain up to 40 ribosomal units. Cell free protein synthesizing systems (cell homogenate as well as reconstituted system) have been prepared and characterized with respect to Mg²⁺, KCI and ATP concentrations, temperature, pH and time course of the reaction. In the cell-free system and in the cellular system the protein biosynthesis is inhibited by chloramphenicol. It is not affected by cycloheximide.     

Protein synthesis in neuronal perikarya isolated from cerebral cortex of the immature rat

Abstract— Homogenates of neuronal perikarya isolated from the cerebral cortex of the 8-day-old rat were incubated with [³H]leucine, and the characteristics of the protein synthetic process were studied. Incorporation of leucine into protein was linear up to 90 min, proceeded optimally at pH 7.6 and was stimulated by K⁺ and NH₄⁺, unaffected by Li⁺ and inhibited by Na⁺. Puromycin, cycloheximide, RNAse, sulphhydryl blocking agents and phospholipase A exerted a pronounced inhibition, whereas chloramphenicol and phospholipase C had no effect. About 42 per cent of the total radioactive protein formed in the optimally fortified in uitro system was recovered in non-sedimentable form. Incorporation into the subcellular fractions of the neuronal perikarya increased steadily with increasing time of incubation. The microsomal fraction acquired the highest specific radioactivity (d.p.m./mg of protein), followed by the mitochondrial and the nuclear + cell debris fractions. The high-speed soluble fraction exhibited the lowest specific radioactivity. Although the addition of L-methionine to a suitably fortified incubation medium inhibited neuronal protein synthesis by about 80 per cent, the addition of D-methionhe, α-methyl-DL-methionine or L-tryptophan was relatively ineffective by comparison.     

Protein synthesis by polysomes from the epidermis of blowfly larvae: Dependence of formation of DOPA-decarboxylase on developmental stage

Polysomes were isolated from the epidermis of six day old larvae and of untanned pupae of Calliphora vicina R.-D. A prerequisite for polysome isolation is that the epidermis be scraped off the overlying cuticle and homogenized in a buffer containing heparin sulfate. A discontinuous sucrose gradient was used to avoid contaminating the preparation with the protein Calliphorin. The incorporation of amino acids into protein by the polysomal preparations has been optimized in respect to monovalent and divalent cations, and the various parameters influencing protein synthesis have been examined. Synthesis is dependent on the presence of pH 5 factors, of ATP and of an energy regenerating system, but not on GTP or an amino acid mixture. Cycloheximide, puromycin, streptovitacin and chloromycetin inhibit protein synthesis. The synthesized polypeptides are not released from the polysomes. One of the proteins synthesized in vitro is DOPA-decarboxylase, as shown by affinity chromatography on a DOPA-decarboxylase IgG-Sepharose column and SDS-acrylamide gel electrophoresis. The polysomes from untanned pupae synthesize three to four times more DOPA-decarboxylase than do those from six day old larvae.     

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.     

Protein synthesis directed by polyadenylated and non-polyadenylated RNA isolated from membrane-bound and free polysomes of Tetrahymena pyriformis

Free and membrane-bound polysomes were prepared from the protozoa Tetrahymena pyriformis using a procedure which gives good recovery and practically no cross-contamination. Polysomes are intact as analysed by sedimentation analysis. Poly(A)-rich RNA and poly(A)-free RNA, isolated from both populations of polysomes, show similar electrophoretic patterns. These RNAs were translated in the rabbit reticulocyte lysate cell-free system and the translation products were analysed by one-dimensional and two-dimensional gel electrophoresis. The most striking differences were found in the two-dimensional electrophoretic analysis namely: (a) a group of polypeptides (10) is synthesized mainly on membrane-bound polysomes, (b) a second abundant group is synthesized mainly in free polysomes (c) and a third class of polypeptides is synthesized on both kinds of polysomes. Poly(A)-free RNAs, isolated from free polysomes, are also able to promote synthesis of some polypeptides. The results are discussed taking into account the fact that T. pyriformis is a non-secretory cell.     

Immunoglobulin synthesis by free polysomes of mouse myeloma cells

Free polyribosomes isolated from mouse myeloma cells in tissue culture synthesize immunoglobulin chains. The presence of these peptide chains in the cytoplasm of intact myeloma cells has been investigated. Some immunoglobulin chains were observed, but it could not be ruled out that these were originally inside cisternae of the endoplasmic reticulum, which were broken during hogenization. We have also investigated the transport of the hypothetical cytoplastic immunoglobulins into the cisternae of the endoplasmic reticulum after incubation with radioactive amino acids and subsequent chase in the absence of protein synthesis. A model to account for synthesis of immunoglobulins on free polysomes is presented. This model assigns specificity for translation on membrane-bound polysomes to the N-terminal region of secretory proteins.     

Inhibition by gamma-hexachlorocyclohexane of acetylcholine-stimulated phosphatidylinositol synthesis in cerebral cortex slices and of phosphatidic acid-inositol transferase in cerebral cortex particulate fractions

• 1 γ-Hexachlorocyclohexane inhibits the ACh-stimulated synthesis of phosphatidylinositol in guinea pig cerebral cortex slices, as measured either by the incorporation of [2-³H]inositol or of ³²P. Phosphatidylinositol synthesis in the control slices is not inhibited.
• 2 The synthesis of phosphatidylinositol from CDP-diglyceride in cerebral cortex microsomal preparations is inhibited by γ-hexachlorocyclohexane. The incorporation of [2-³H]inositol into lipid in the absence of added cytidine nucleotide in these preparations is not inhibited.
• 3 δ-Hexachlorocyclohexane profoundly inhibits phosphatide synthesis and phosphate metabolism in cerebral cortex slices both in the presence and absence of ACh. This isomer also inhibits the exchange reaction for the incorporation of [2-³H]inositol into lipid in the microsomal preparations.
• 4 α-, and β-Hexachlorocyclohexanes do not inhibit either ACh-stimulated or control synthesis of phosphatidylinositol in cerebral cortex slices; nor do they inhibit the exchange reaction for [2-³H]inositol incorporation into lipid in the microsomal preparations.
• 5 The specific effects of γ-hexachlorocyclohexane are taken as providing evidence that ACh-stimulated phosphatidylinositol synthesis in cerebral cortex slices probably involves the CDP-diglyceride pathway. The possibility is discussed that the primary action of ACh in this system is to cause an increased activity of diglyceride kinase to provide phosphatidic acid for this pathway.

     

Protein synthesis with membrane-bound polysomes and albumin messenger RNA from livers of mutant mice

Summary Investigation of deficiencies in serum protein synthesis resulting from deletion-mutations at the albino locus in mice was continued usingin vitro conditions. Previous work showed that although total protein synthesis was only slightly lower in livers from albinos, newly synthesized protein appearing in plasma was 22% of that in controls. It was thought that the disorganized endoplasmic reticulum and Golgi apparatus, characteristic for the liver (and kidney) of these mutants, might be responsible for the observed deficiencies. In the present study mebrane-bound polysomes isolated from the livers of newborn albinos were 55% (c^3H/c^3H strain) and 62% (c^14CoS/c^14CoS strain) as efficient as those from normal littermates in incorporating radioactive leucine into protein in a cell-free system. These differences could not be eliminated by the addition of excess liver mRNA, exogenous soluble factors or by the exchange of cell sap between albino and control polysomes. In an earlier study albino liver slices synthesized only 13% (or 17% per mg of total protein synthesized) as much albumin as controls. We have now found that the level of albumin poly(A)⁺-RNA isolated from albino livers and assayed with a reticulocyte lysate, was almost as high (85%) as in controls. It was concluded that the very low level of albumin synthesis in albino livers did not result from a deficiency of albumin mRNA. Whether the rate-limiting step in synthesis of albumin in mutant livers is at the level of translation or processing for secretion requires further investigation.     

Uptake and effects of melatonin on the synthesis of proteins by the rat cerebral cortex

Slices of rat parietal cerebral cortex took up and retained [³H] melatonin up to a tissue concentration about 4-fold to that present in the incubation medium. This phenomenon was time-dependent, maxima being observed after 180 min-incubations Eighty to 93% of the radioactivity present in the cerebral cortex slices was chromatographically identified as melatonin. Even at the highest melatonin concentration that could be dissolved in the incubation media, a constant proportion of [³H] melatonin was bound to cortical slices, indicating that within this concentration range, melatonin binding is independent of its concentration. Melatonin effects on protein synthesis in the rat cerebral cortex were investigated by studying the incorporation of [³H] L-leucine into proteins in cerebral cortex of rats injected s.c. with 10 or 100 μg/day of melatonin for 5 to 10 days. Both treatments caused leucine incorporation into proteins to increase significantly by about 50 to 60%.     

Regulation of protein synthesis in Tetrahymena: isolation and characterization of polysomes by gel filtration and precipitation at pH 5.3.

The fraction of ribosomes loaded on polysomes is about 95% in logarithmically growing Tetrahymena thermophila, and about 4% in starved cells. Cytoplasmic extracts from cells in these two physiological states were used to develop column chromatographic methods for the purification of polysomes. Bio-Gel A 1.5 m was found to separate total cytoplasmic ribosomes from many soluble proteins, including RNAse, with no detectable change in the polysome size distribution. Polysomes can be separated from monosomes and non-polysomal mRNA by chromatography on Bio-Gel A 15 m without size selection. These methods can easily be adapted to large scale preparations of polysomes, even from cells where a small fraction of the ribosomes is on polysomes. A method is described for reversible precipitation of polysomes and monosomes from dilute solutions at pH 5.3 which greatly facilitates polysome isolation. Hybridization of 3H-labeled polyU to RNA isolated from column fractions has been used to demonstrate that purification of EDTA released polysomal mRNA can be performed using the column chromatography procedures described here. These methods have been employed to demonstrate that most of the cytoplasmic mRNA in log-phase Tetrahymena is loaded onto polysomes while most of the mRNA is starved cells exists in a non-polysomal form.     

Further studies on the cell-free synthesis of procollagen-collagen by chick embryo polysomes

Free and membrane bound polysomes were prepared from 8-day-old chick embryos. Both polysome preparations were equally active in protein synthesis but procollagen-collagen synthesis was carried out exclusively by the membrane bound polysomes. The collagenous product was analyzed by DEAE-cellulose chromatography and after hydroxylation with peptidyl proline hydroxylase had a hydroxyproline/proline ratio of 0.77. This suggests that the collagenous product synthesized by the membrane bound polysomes is procollagen.