Evidence that free polysomes are not the precursors of membrane-bound polysomes in rat liver

We tested, in rat liver, the postulate that free polysomes were precursors of membrane-bound polysomes. Three methods were used to isolate free and membrane-bound ribosomes from either post-nuclear or post-mitochondrial supernatants of rat liver. Isolation and quantitation of 28 S and 18 S rRNA allowed determination of the 40 S and 60 S subunit composition of free and membrane-bound ribosomal populations, while pulse labeling of 28 S and 18 S rRNA with [6-¹⁴C]orotic acid and inorganic [³²P]phosphate allowed assessment of relative rates of subunit renewal. Throughout the extra-nuclear compartment, 40 S and 60 S subunits were present in essentially equal numbers, but, free ribosomes contained a stoichiometric excess of 40 S subunits, while membrane-bound ribosomes contained a complementary excess of 60 S subunits. Experiments with labeled precursors showed that throughout the extra-nuclear compartment, 40 S and 60 S subunits accumulated isotopes at essentially equal rates, however, free ribosomes accumulated isotopes faster than membrane-bound ribosomes. Among free ribosomes or polysomes, 40 S subunits accumulated isotopes faster than 60 S subunits, but, this relationship was not seen among membrane-bound ribosomes. Here, 40 S subunits accumulated isotope more slowly than 60 S subunits. This distribution of labeled precursors does not support the postulate that free polysomes are precursors of membrane-bound polysomes, but, these data suggest that membrane-bound polysomes could be precursors of free polysomes.     

Membrane-bound ribosomes in kidney: methods of estimation and effect of compensatory renal growth

Membrane-bound ribosomes are thought to secrete protein for export and free ribosomes to secrete protein for intracellular use. The proportion of the total ribosomes that is bound to membranes in normal mouse kidneys has been estimated by three different methods, and the results have been compared with those obtained by a fourth method used by us previously. The most valid estimates appear to be those obtained (a) by comparison of radioactivity in peaks representing free and membrane-bound ribosomes on linear sucrose gradients after labeling for 24 hr with ¹⁴C-orotic acid, and (b) by measurements of optical density in free and bound ribosomes that had been separated by centrifugation on discontinuous gradients of 0.5 M/2.0 M sucrose. Analyses by these methods show that about 20–25% of the ribosomes in a postnuclear supernatant prepared from mouse kidneys, but only 10–15% of the ribosomes in a post-mitochondrial supernatant, are membrane-bound. About 75% of the bound ribosomes sediment as polysomes of many different sizes. The proportion of membrane-bound ribosomes and their aggregation into polysomes were unchanged in kidneys undergoing compensatory hypertrophy after removal of the opposite kidney. These experiments show that, unlike liver, kidney has a predominance of free ribosomes compared to bound ribosomes; those ribosomes that are membrane-bound do not become free during compensatory renal growth.     

Protein metabolism. 3. Relationship between albumin metabolism and elevated liver protein synthesis in the guinea pig infected with Trichostrongylus colubriformis

Studies of the incorporation of ¹⁴C-l-leucine into polypeptides by isolated liver ribosomes from guinea pigs confirmed previous in vivo studies that showed that Trichostrongylus colubriformis infection results in an elevated hepatic protein synthesis.The increased rate of protein synthesis was associated with the membrane-bound ribosomes that synthesize the circulating plasma proteins. Inappetance of infected animals was not resposible for the increased rate of synthesis by the membrane-bound ribosomes, but it was found that undernourishment may stimulate synthesis by free ribosomes.Plasma albumin turnover rate and loss into the intestine were both significantly increased in infected guinea pigs. It was concluded that these changes stimulated protein synthesis by membrane-bound ribosomes.The importance of elevated liver protein synthesis and loss of plasma protein in gastrointestinal nematode infections is discussed.     

The importance of membrane-bound ribosomes during the activation of thymocytes by concanavalin A.

Concanavalin A (ConA) seleclively enhanced the incorporation of [³H]leucine into a range of proteins of thymocytes incubated in vitro. At the same time ConA seemed to selectively enhance the synthesis of proteins that occurred on membrane-bound ribosomes (extracted from the mitochondrial fraclion with 1% Triton X-100 buffer). The protein synthetic ‘commitment’ of ribosomes was assessed from the stability of ribosomes in 500 mM KC1 before and after puromycin treatment. This indirect method was necessary because of some polysome degradation in the case of membrane-bound ribosomes. Membrane-bound ribosomes were found to be more than 3 times as ‘committed’ as were free ribosomes and ConA increased their commitment by 37–54%. These observations indicate the potential importance of membrane-bound ribosomes in the regulation of thymocyte protein synthesis, particularly during ‘antigenic’ activation, even though this ribosome fraction constituted less than 20% of the total ribosome population.     

Free and membrane bound ribosomes of the cotyledons of Vicia faba (L.)

Summary Changes in the quantities of free and membrane-bound ribosomes were followed in the cotyledons of developing seeds. During the phase of storage protein synthesis, free and membrane bound ribosomes do not interchange, and as both classes synthesise protein in vivo, it is possible that they may synthesise different groups of proteins. This suggestion is discussed in relationship to the developing cotyledon.     

Effect of Aflatoxin B1 and Corticosterone on Ribosome-distributions between Free and Membrane-bound States and between Monosomes and Polysomes in Mouse Liver

The aim of this research was to examine the inhibitory effect of aflatoxin B₁, one of the most potent hepatocarcinogen, on the translational step in mouse liver. It has been shown that polysomes were released in vitro from microsomal membrane prepared from rat liver by incubation with aflatoxin B₁ and that this release of ribosomes was prevented by addition of corticosterone in the incubation medium.

In this paper, the same phenomenon was proved to occur in vivo by an improved fractionation methods, in which ribosome-distributions can be analyzed quantitatively, not only between free and membrane-bound states but also between monosomes and polysomes. Administration of aflatoxin B₂ to mice induced reductions of membrane-bound ribosomes and polysomes, with concomitant increases of free ribosomes and monosomes in liver. Simultaneous administration of corticosterone prevented this alteration of ribosome-distributions.

From these results, it was deduced that a release of polysomes from membrane occurred primarily by administrating aflatoxin, which then caused a shortening of half-life of mRNA on polysomes, resulting in an increase of the amount of monosomes.     

A method for visualizing and quantifying the total complement of free and membrane-bound ribosomes in rat liver.

A method is described for both visualization and quantification of the total complement of rat liver free and membrane-bound ribosomes, undegraded by nucleolysis and unaggregated by pelleting. The method involves: (a) differential centrifugation of liver homogenate which separates free and membrane-bound ribosomes; (b) treatment of the fractions with detergents to solubilize membranes and remove nuclei; (c) centrifugation of a portion of each fraction to remove all the ribosomes; (d) sedimentation of the samples and blanks on sucrose gradients; and (e) difference photometric scanning of the gradients, sample minus ribosome-free blank, to detect the ribosomes free of interference from nonribosomal materials. The use of the SW 56 rotor in the initial centrifugation and of a high Mg²⁺ concentration (20 mm) in the medium used to suspend the bound fraction prior to detergent treatment were found to be essential in obtaining bound polysomes of large size (～19-somes). The difference scanning technique is shown to be a sensitive, accurate, and reproducible means of eliminating interference from nonribosomal materials, principally detergents and protein, and of quantifying ribosomes in both fractions. The method is rapid (3.5 h), simple to perform, and well suited for the analysis of multiple liver samples. It can be used to assess the concentration, distribution, organization, and average size of the total complement of rat liver free and membrane-bound ribosomes in a single experiment.     

In vitro exchange of ribosomal subunits between free and membrane-bound ribosomes

Studies on the distribution of isotopieally labeled ribosomal subunits between free and membrane-bound ribosomes from rat liver showed that, upon release of nascent polypeptides in vitro, the small subunits of membrane-bound ribosomes could exchange with small subunits derived from free polysomes. However, under the same conditions, the large subunits of membrane-bound ribosomes did not exchange efficiently with large subunits derived either from free or bound polysomes; instead, the addition of large subunits caused a transfer of microsomal small subunits into a newly formed pool of free monomers.The small subunit exchange required a macromolecular fraction of the cell sap, was stimulated by ATP or GTP, and occurred at low concentrations of magnesium ions.Sodium dodecyl sulfate, polyacrylamide gel electrophoresis revealed close similarities between the protein complement of subunits from free and membrane-bound ribosomes, with the exception of one protein band which was more intense in free large subunits.     

Ribosome metabolism in hormone-treated Jerusalem artichoke tuber slices in the absence and presence of 5-fluorouracil

In order to examine the relation of protein synthesis to the onset of growth, changes in ribosome content and activity were compared in aged, metabolically active Jerusalem artichoke (Helianthus tuberosus L.) slices incubated in water or 2,4-dichlorophenoxyacetic acid+kinetin. In water, cells do not grow or divide and rRNA and protein levels remain constant. The percentage membrane-bound (mb) ribosomes drops from 25% to 16% during 24h. At the same time the proportion of ribosomes active in protein synthesis in both free and mb populations declines from about 69% to 54%. In auxin+kinetin, cell expansion occurs and is accompanied by a 3-fold increase in rRNA and a 50% increase in total protein content. The percentage mb ribosomes remains at 25% throughout 48 h of growth. During the first 24h of growth 70% of ribosomes in both free and mb populations are active; this value declines to near water levels at 48 h. Considering the large increase in total ribosomes the number of synthetically active ribosomes is substantially increased during growth. 5-Fluorouracil (5-FU) does not inhibit hormone induced growth but does depress total rRNA content by about one-third. It also reduces [³H]uridine incorporation into ribosomes by 70% and the newly made ribosomes are mostly inactive in protein synthesis. On the other hand, the inhibitor does not significantly affect the proportion of total ribosomes active in protein synthesis and only partially reduces protein accumulation during the second 24 h of growth. It is suggested that while ribosome production is reduced in 5-FU, ribosome turnover is also retarded resulting in retention of near normal capacity for protein synthesis and growth.     

Ribosomes in the lutoïd fraction (=lysosomal compartment) from Hevea brasiliensis Künth. (Mull.-arg.) latex

Lutoïd ribosomes represent 11.9% of the total ribosomal content of latex from Hevea brasilensis. Their two high-molecular-weight RNA components are identified. When undegraded, their molecular weights are calculated to be 1.3×10⁶ and 0.7×10⁶. Their nucleotide base composition is determined. The occurrence of lutoïd membrane-bound ribosomes and the lability of the 1.3×10⁶RNA component are discussed in relation to the biological role of lutoïds, more generally of a lysosomal compartment in the cellular function.     

Distinctions between rabbit lymph node cell populations responding to primary and secondary injections of keyhole limpet hemocyanin

Rabbits were injected once or twice into the hind foot pads with alum-precipitated keyhole limpet hemocyanin. At the height of the primary or secondary responses individual rabbits were sacrificed for the preparation of lymph node cell suspensions from the regional lymph nodes. These cells were employed for the in vitro study of antibody synthesis by the incorporation of ¹⁴C-leucine and the secretion of antibody by the time of appearance of radioactive antibody in the medium. The primary response cells rapidly synthesized and secreted IgM and IgG antibodies. The secondary response cells rapidly synthesized IgM and IgG antibodies, secreted IgG antibody promptly, but secreted the IgM antibody with a lag of six to ten hours. Microsomal fractions could not be prepared from the primary response cells, but were readily produced from the cells of the secondary response. The primary response cells contained mainly free ribosomes, those of the secondary response predominantly membrane-bound ribosomes. It was postulated that IgM antibody was not secreted until it was glyco-sylated in the Golgi apparatus and that the lag in secretion entailed the time for this rate-limiting step to occur.     

Partial characterisation of and the effect of insect growth hormones on the ribosomes and polyribosomes of the nematode, Panagrellus redivivus

An investigation of some of the properties of the ribosomes and polyribosomes of Panagrellus redivivus revealed that: the polyribosomal RNA was resolvable into eight species, four of which possessed typical S-values and M.W.s and closely resembled those of Aphelenchus avenae; the estimated S-value of the ribosomes was 92; the polyribosomes were mainly free and not membrane-bound: and, the polyribosomes showed a low level of activity in in vitro amino-acid incorporation. The polysomes (double-labelled or unlabelled) revealed no effect of synthetic juvenile hormone or ß-ecdysone (1 × 10^?5M) on their polyribosomal profile, at intervals up to and including 19 h of incubation.     

Membrane-associated protein synthesis of mammalian cells. II. Isopycnic separation of membrane-bound polyribosomes

The membrane-bound ribosomes of HeLa cells were analyzed in CsCl buoyant-density gradients. There exist two classes: a heavy class with a density of 1.55 g/ cm³ and a light class heterogeneously distributed with a mean density of 1.49 g/cm³. The lower density of the light class is due to an increased ratio of protein to RNA. This additional protein is not removed with increased ionic strength. Both classes of ribosomes incorporate amino acids at a rate comparable to non-membrane-bound ribosomes. The heavy and light ribosomes appear to correspond to the EDTA-sensitive and the EDTA-resistant ribosomes, respectively.     

Age dependent changes in fungi: Ribosomes and protein synthesis inRhizoctonia solani mycelium

Summary Increasing age ofRhizoctonia solani cells was accompanied by a decrease in protein synthesis but not by a fall in the number of ribosomes present. There was, however, a shift from predominantly polyribosomes in young cells actively synthesizing protein, to mainly monoribosomes in older less active cells, and it is suggested that protein synthesis is restricted in these older cells by some defect at the initiation step of protein synthesis. The major site of protein synthesis throughout ageing was the free ribosome fraction with little or no contribution from membrane-bound ribosomes. For reasons not understood, the free ribosomes failed to sediment through 2.0 M sucrose, and only by using 1.4 M sucrose were good separations obtained.     

Interaction between glutathione and the endoplasmic reticulum in cyclic protein synthesis in sea urchin embryos.

Interaction between an oxidoreduction system and cyclic protein synthesis was studied in sea urchin embryos. When assayed enzymatically, in both in vivo and in vitro systems, the contents of GSH and GSSG varied inversely in a cyclic fashion. Diamide at 0.5 mM inhibited amino acid incorporation in not only the cyclic phase but also the basal phase, but 4-nitroquinoline-N-oxide at 1 μM inhibited only the cyclic phase. Sea urchin embryos contained membrane-bound ribosomes, and pulse-labeling with amino acids suggested that free ribosomes were responsible for the basal phase and membrane-bound ribosomes were responsible for the cyclic phase of amino acid incorporation. Thiol-disulfide interchanging enzyme was found in the endoplasmic reticulum fraction. An extract of the endoplasmic reticulm caused stimulation of binding of acetylphenylalanyl-tRNA to 40S ribosomes and polyphenylalanine synthesis in the presence of low GSH concentrations. An extract of the endoplasmic reticulum also catalyzed oxidoreduction from GSH to the KCl-soluble protein. Thus, the periodic stimulation of protein synthesis is interpreted to be the result of the periodic activation of membrane-bound ribosomes by the thiol-disulfide interchanging enzyme which accepts selectively the signal from the GSH cycle.     

Ribosomes and Polysomes in Cucumber Leaves during Growth and Senescence

The quantity of RNA in the ribosomal fraction of the first leaf of cucumber (Cucumis sativus) increases during growth, reaches a maximum before the final fresh weight is attained, and then decreases. The main changes are in the free ribosome fraction, the quantity of membrane-bound ribosomes remaining about constant. Few 65.5S chloroplast ribosomes are present in small leaves; however, they increase in quantity rapidly during growth and form about half of the ribosomes present in the mature fully green leaf. The cytoplasmic ribosomes have a sedimentation coefficient of 77.6S. Ribonuclease-sensitive polysomes were present in leaves of all ages except possibly the very oldest. The proportion of ribosomes in polysome form decreases during growth and then remains roughly constant during senescence. Following maturation of the leaf, the rate of incorporation of ³²P into ribosomal-fraction RNA begins to decline. This decline could account for the loss of ribosomes during the early stages of senescence. The possibility that leaf ribonuclease might be responsible for the final, more rapid loss of RNA, is discussed.     

Characterization of poly(a)-protein complexes isolated from free and membrane-bound polyribosomes of Ehrlich ascites tumor cells

Proteins present in messenger ribonucleoprotein particles were labeled with [³⁵S]-methionine in Ehrlich ascites tumor cells in which synthesis of new ribosomes was inhibited. Poly(A)-protein complexes were isolated from free and membrane-bound polyribosomes by sucrose gradient centrifugation and affinity chromatography on oligo(dT)-cellulose. Both classes of Poly(A)-protein particles contain a poly(A) chain of about 70 adenyl residues and a protein with a molecular weight of 76000 attached to it.     

Distribution of membrane-bound and free ribosomes in growing yeast.

1. The distribution of membrane-bound and free ribosomes was investigated in stationary as well as in growing yeast cells. the relative amount of free ribosomes varies with the growth phase of the cell culture. During the duplication phases of the cell, relative maxima of free ribosomes can be found. However, the absolute amount of free ribosomes is fairly constant during the growth of the cells. 2. Membrane-bound ribosomes show lower polypeptide synthesis activity in a cell-free, poly (U)-dependent system than free ribosomes. 3. There is no difference in the distribution pattern of free and membrane-bound ribosomes in growing yeast cells of different ploidy. 4. A turnover between free and membrane-bound ribosomes is suggested to be in agreement with the hypothesis of Branes and Pogo ((1975) Eur. J. Biochem. 54, 317-328).     

Surface topography of the Bacillus stearothermophilus ribosome.

Summary The surface topography of the intact 70S ribosome and free 30S and 50S subunits from Bacillus stearothermophilus strain 2184 was investigated by lactoperoxidase-catalyzed iodination. Two-dimensional polyacrylamide gel electrophoresis was employed to separate ribosomal proteins for analysis of their reactivity. Free 50S subunits incorporated about 18% more ¹²⁵I than did 50S subunits derived from 70S ribosomes, whereas free 30S subunits and 30S subunits derived from 70S ribosomes incorporated similar amounts of ¹²⁵I. Iodinated 70S ribosomes and subunits retained 62–78% of the protein synthesis activity of untreated particles and sedimentation profiles showed no gross conformational changes due to iodination. The proteins most reactive to enzymatic iodination were S4, S7, S10 and Sa of the small subunit and L2, L4, L5/9, L6 and L36 of the large subunit. Proteins S2, S3, S7, S13, Sa, L5/9, L10, L11 and L24/25 were labeled substantially more in the free subunits than in the 70S ribosome. Other proteins, including S5, S9, S12, S15/16, S18 and L36 were more extensively iodinated in the 70S ribosome than in the free subunits. The locations of tyrosine residues in some homologus ribosomal proteins from B. stearothermophilus and E. coli are compared.