The effect of insulin on proteins associated with free,cytoskeletal-bound and membrane-bound polysome populations.

It is known that insulin treatment increases the rate of protein synthesis in many cells and tissues and that it causes changes in the distribution of ribosomes between free (FP), cytoskeletal-bound (CBP) and membrane-bound polysome (MBP) populations. This paper concerns an analysis of the pattern of proteins in high-salt extracts of FP, CBP and MBP isolated from Krebs II ascites and MPC-11 cells. A combined detergent/salt extraction procedure was used to isolate the three fractions of polysomes from control cells and from cells following short-term stimulation with insulin. There were differences in the protein patterns in the individual fractions and changes occurred after insulin stimulation.     

Free,cytoskeletal-bound and membrane-bound polysomes isolated from MPC-11 and Krebs II ascites cells differ in their complement of poly(A) binding proteins

A three-step detergent/salt extraction procedure (Vedeleret al., Mol Cell Biochem 100: 183–193, 1991) was used to isolate free polysomes (FP), cytoskeletal-bound polysomes (CBP) and membrane-bound polysomes (MBP) from MPC-11 and Krebs II ascites cells. Polysomes were pelleted, washed with high salt buffer and re-pelleted. Proteins in the dialysed high-salt extracts were subjected to poly(A) Sepharose chromatography and poly(A) binding and non-binding proteins were separated by SDS-PAGE. In MPC-11 cells the FP fraction contains thirteen poly(A) binding proteins and four non-poly(A) binding proteins while the corresponding fraction in Krebs II ascites cells has four poly(A) binding proteins and six proteins which do not bind poly(A). The CBP fraction isolated from MPC-11 cells has a complement of ten poly(A) binding proteins, four which are non-poly(A) binding, and a protein of 105 kDa which has both poly(A) binding and non-poly(A) binding properties. In the CBP fraction prepared from Krebs II ascites cells a protein band at 32 kDa exhibits both poly(A) binding and non-poly(A) binding properties. In this fraction there are six poly(A) binding proteins and an additional eight which do not bind poly(A). Of the total number of proteins eight of these have a molecular weight below 40 kDa. The MBP fraction in MPC-11 cells contains three poly(A) binding proteins and eleven with non-poly(A) binding properties. In contrast this fraction in Krebs II ascites cells has a complement of thirteen poly(A) binding and ten non-poly(A) binding proteins. The results show differences in the poly(A) binding properties of the proteins in the three polysome fractions and that the complements of polysome-associated proteins are different in the two cell lines. This may be related to the differences in the growth characteristics of MPC-11 and Krebs II ascites cells.     

Possible involvement of the cytoskeleton in the regulation of barley caryopsis dormancy and germination

This study was conducted on barley cv. Ars. caryopses collected at full ripeness and divided into two batches. From one batch (dormant caryopses) polysomes were isolated from embryos immediately after harvesting and after two days of germination. From the other batch (non-dormant caryopses) the same was done after eight months storage in a dry state. A low ionic strength cytoskeleton-stabilizing buffer was used for the isolation of polysomes. Four different fractions of polysomes were examined: free polysomes (FP), membrane-bound polysomes (MBP), cytoskeleton-bound polysomes (CBP) and cytoskeleton-membrane-bound polysomes (CMBP). In germs grown from non-dormant caryopses, the first two fractions (FP + MBP) made up about 78 % of the total ribosomal material, whereas in embryos of dormant, imbibed caryopses, two last fractions (CBP + CMBP) made up about 71 %. The percentage of polysomes after 48 hours of imbibition of dormant caryopses in the FP, MBP and CBP was only about 13 % (i.e., 87 % monosomes), whereas a greater proportion (19.4 %) was found in the CMBP. The highest incorporation of ³H-uridine and ¹⁴C-amino acids (after 48 hours of germination and 0.5, 3 and 6 hrs incubation with precursors) took place in trhc CMBP both in dormant and non-dormant caryopses The major amount of the two polysome fractions associated with the cytoskeleton (CBP and CMBP) and the higher activity of CMBP in protein synthesis in embryos of dormant, imbibed triticale caryopses may indicate a significant role for polysomes associated with the cytoskeleton in the control of protein synthesis in dormant and germinating caryopses.     

Significant role for polysomes associated with the cytoskeleton in the control of protein synthesis during germination of triticale caryopses in the presence of abscisic acid

The influence of abscisic acid (ABA) on the process of polysome formation and synthesis of newly-formed proteins by different polysome populations was studied. Triticale caryopses were germinated in water or various ABA concentrations for 48 hrs, and afterwards they were transferred to a solution of ¹⁴C-amino acids and germinated for an additional 30 min. Embryos were separated from caryopses, and four polysome populations were isolated: the FP (free polysomes), MBP (membrane-bound polysomes), CBP (cytoskeleton-bound polysomes) and CMBP (cytoskeleton-membrane-bound polysomes). ABA retarded both the process of polysome formation and their activity in forming new proteins in vivo in all studied fractions. Participation of polysomes in total ribosomal materials (sub-units, monosomes and polysomes) of each polysome population in the control sample was as follows: FP — 77; MBP — 72; CBP — 70 and CMBP — 66 %, whereas in sample treated by ABA (100 μM) it was accordingly: 17; 23; 27 and 28%. The largest population made up FP (in control sample 69%), participation of MBP was always lower and ranged from about 19 to 30 %. Participation of polysome populations bound with the cytoskeleton CBP and CMBP, both in control sample as well as in samples treated with 1 and 10 μM ABA solution, was only a few per cent. It should be noted that when the ABA concentration was higher (100 μM) (process of germination was strongly inhibited), participation of those two populations (CBP and CMBP) was much increased in embryos, respectively to about 18 and 20 %. In both the control group and in embryonal tissue treated with ABA increasing incorporation of radioactive precursors to newly-formed proteins in vivo in fractions of polysomes isolated by following buffers: C (FP), C + PTE (MBP), C + Tris (CBP) and buf. U (CMBP) was observed. It should be noted, that the biggest incorporation of ¹⁴C-amino acids into nascent polypeptide chains was found in the last polysome population (CMBP). In the sample treated with ABA (100 μM) the activity of this fraction (CMBP) in forming new proteins is several times, and in the case of FP dozens of times, more intense. Increased participation of CBP and CMBP in embryos of triticale caryopses treated with ABA (100 μM) and the largest incorporation of ¹⁴C-amino acids into nascent polypeptide chains synthesised by CMBP, may indicate the important role of proteins formed by polysomes associated with cytoskeleton in inhibition of germination and seedling growth by ABA.     

Polysome formation and stability in pea stem and root tissue

Polysome stability and the formation of various polysomal populations in pea stem and root tissue were examined. Both total ribosomal fraction and four polysome populations were isolated: FP (free polysomes), MBP (membrane-bound polysomes), CBP (cytoskeleton-bound polysomes) and CMBP (cytoskeleton-membrane-bound polysomes). The content of above mentioned populations decreased in roots and stems during germination. In both roots and stems a gradual decrease of FP participation in the total polysomal population was also observed during germination. On the other hand, an obvious increase in participation of CMBP population in the total polysomes pool was observed in later stages of germination. Increase of CMBP participation in pea root and stem tissues in later stages of germination is probably due to intensive enzymatic protein synthesis taking place in them. These proteins may participate in elongating growth of cells. The results of investigation on polysomes stability showed that total polysomes isolated from pea roots appeared to be more resistant to digestion by exogenous ribonuclease (EC 3.1.27.5) than polysomes isolated from stems. As protein-mRNA interactions are widely known and ribosomes are also very adhesive structures, numerous non-ribosomal proteins are present in the polysome preparations. We suppose that changes in proteins bound to polysomes indicated by us previously, significantly influence both the stability and also translatability of polysomes isolated from different plant organs.     

Importance of cytomatrix-bound polysomes to synthesis of lysine-containing proteins in triticale germs under ABA treatment

The influence of exogenous abscisic acid (ABA) on the content of free polysomes (FP), membrane-bound polysomes (MBP), cytoskeleton-bound polysomes (CBP) and cytomatrix-bound polysomes (CMBP) in triticale germs as well as in vitro protein synthesis by these four polysomal fractions were studied. During translation, proteins were biotinylated for chemiluminescence detection. We have found that ABA changed both the content of FP, MBP, CMP and CMBP in germ tissue, and their subsequent translation activity. At 100 μM ABA, the content of FP and MBP was over fourfold lower compared to the control, whereas the amounts of CBP and CMBP were about two- and threefold higher, respectively. Moreover, the estimation of the share of polysomes in each ribosomal fraction (sub-units, monosomes, polysomes) showed that, at 100 μM ABA, cytomatrix-bound polysomes, which constituted 90% of polysomes, were the predominant class in ABA-treated germs while membrane-bound polysomes, which made up 82% of polysomes, dominated in the control. A high level of CMBP in ABA-treated tissues may indicate that this class of polysomes participates in ABA-induced synthesis of proteins. In turn, the inhibition of MBP under ABA-treatment is probably due to the delayed protein synthesis which takes place on these polysomes. We identified two lysine-containing proteins synthesized on both of the above classes of polysomes, whose synthesis was altered due to ABA application. Synthesis of a 47 kDa protein on MBP was inhibited, while synthesis of a 79 kDa protein on CMBP is strongly enhanced by ABA influence. The importance of these findings is discussed.     

Effect of osmotic stress on the formation of a population of polysomes and their stability in pea (<Emphasis Type="Italic">Pisum sativum</Emphasis> L.) seeds

Plants growing under natural conditions are constantly exposed to various stress factors, which can restrain their productivity and limit yields. This paper deals with the effect of long- and short-term osmotic stress followed by recovery on the formation of polysomes and their stability during germination of pea (Pisum sativum L.) seeds. By isolating polysomes, it is possible to obtain an index which evidences the ability of tissues to synthesize proteins. Changes in the distribution of polysomes often precede measurable changes in amounts of proteins. Under osmotic stress, the dominant population of polysomes was the population of free polysomes (FP). The share of membrane-bound polysomes (MBP) and cytoskeleton-bound polysomes (CBP) and cytoskeleton-membrane-bound polysomes (CMBP) in the total fraction of ribosomes increased under intensive (−1.0 and −1.5 MPa) osmotic stress. These results can suggest that the bound forms of polysomes play an important role in the synthesis of stress proteins. In addition, the stability of polysomes isolated from pea early seedlings growing under unstressed control and osmotic stress conditions was tested. It turned out that polysomes formed under osmotic stress conditions (especially the CMBP) were more resistant to the activity of exogenous ribonucleases than the polysomes in the control samples. Under stress conditions it is highly likely that ribosomes become more densely packed on mRNA thus making it more resistant to ribonuclease. This is just one of the many mechanisms regulating stability of mRNA.     

The influence of abscisic acid on different polysomal populations in embryonal tissue during pea seeds germination

The influence of abscisic acid (ABA) on the processes of formation of different polysomal populations, their structures and stability in embryonal tissue during pea seeds germination was studied. The contents of total ribosomal fraction increased in all samples up to 72 h of germination and then decreased. The contents of polysomal population (FP, MBP, CBP and CMBP) extracted from the embryonal tissue after 72 hrs of germination of pea seeds were then quantified. It turned out that in examined tissue of control sample, fraction of free polysomes (FP) was the most abounded. This population of polysomes in sprouts decreased after ABA treatment. FP content decreased even more when the higher ABA concentration was applied during germination. Similar changes were observed in the fraction of membrane-bound polysomes (MBP). Quite different tendencies were found, however, in forming population of the cytoskeleton-membrane-bound polysomes (CMBP). The CMBP population content in embryonal tissue increased in a dosage dependent manner with increasing concentration of ABA applied during seed germination. This indicates the important role of CMBP fraction in synthesis of specific proteins in embryos in the time when processes of seeds germination are retarded by ABA. In the final part we examined the stability of polysomes isolated from sprouts of germinating seeds in water and sprouts isolated from seeds treated with ABA (100 μM) during germination. Total polysomes isolated from embryonal tissue of germinating seeds treated with ABA showed much higher resistance to exogenous ribonuclease digestion than total polysomes of control sample. The obtained results suggest that ABA influence on different polysomal population formation also controls their stability.     

Effects of serum deprivation, insulin and dexamethasone on polysome percentages in C2C12 myoblasts and differentiating myoblasts.

An increase in the rate of protein synthesis in living cells can be achieved by regulating the quantity of mRNA, ribosomes, and enzymes available for translation or by regulating the efficiency at which existing components are used. Efficiency can be measured by comparing the number of ribosomes actively engaged in the synthesis of protein (polysomes) to the pool of free ribosomes. The objective of this study was to determine the percentage of ribosomes found as polysomes in C2C12 cells deprived of serum or exposed to insulin or dexamethasone 24 h before and after being stimulated to differentiate. Individual 60 mm culture dishes were exposed to serum-free control medium, medium containing serum, insulin, or dexamethasone for a period of 1 h or 2 h and then quickly frozen. The ribosomes and polysomes from these cells were separated by ultracentrifugation on 15 to 60% sucrose gradients and the absorbance across the gradient at 254 nm was recorded. Polysome percentages were determined as the area under the polysome peak divided by the total area under the curve. Serum deprivation caused a 12% decline in the percentage of ribosomes found as polysomes (P < 0.01). Dexamethasone caused a quadratic decline (P < 0.05) in polysome percentage, while insulin yielded a quadratic increase (P < 0.05). Protein synthesis assays measuring 3H-tyrosine uptake showed similar responses. These changes occurred in the absence of any differences in total RNA concentration. It was concluded that differentiation and the absence of serum in the media reduced the rate of recruitment of ribosomes for protein synthesis. Insulin increased ribosome recruitment which was also observed by a similar increase in incorporation of radio-labeled tyrosine.     

Lipogenesis in rat tissues following carbohydrate refeeding: Spleen lipogenesis is modulated by insulin

Intraperitoneal administration of [1,2-¹⁴C]-acetate to Wistar rats was used to assess tissue lipogenic rates after estimating the incorporation of the label into the tissular lipid fractions. Refeeding the animals with glucose (after an overnight fast) induced an increase in white adipose tissue (4.5 fold), liver (4.1 fold), small intestine (1.9 fold), carcass (2.9 fold) and spleen (3.7 fold) lipogenesis (expressed as the radioactivity present in the lipid fraction corrected by the plasma circulating radioactivity). No changes were found following refeeding in either brain or brown adipose tissue. Administration of mannoheptulose (an inhibitor of insulin secretion) to refed rats completely abolished the increased lipogenesis in white adipose tissue, liver, carcass, spleen and small intestine, thus suggesting that insulin secretion is involved in this phenomenon. This is the first report showing that spleen lipogenesis may be modulated by refeeding via insulin secretion and suggests an important role of this organ on the in vivo lipogenic response of the organism after carbohydrate refeeding. (Mol Cell Biochem 175: 149–152, 1997)     

Mechanism of protein synthesis inhibition during stationary phase in Bacillus stearothermophil US

Summary The appearance of a protein (association factor I) in ribosomes from Bacillus stearothermophilus at stationary phase of growth is described. Association factor I is present on 30S subunits and 30S–50S ribosomal couples, but not on 50S subunits. This protein is responsible for the low levels of polyphenylalanine synthesis shown by stationary phase ribosomes. Association factor I is able to bind to free 30S–50S ribosomal couples but not to polysomes, and exerts its effect by inhibiting the initiation step of protein synthesis. Ribosomes preincubated with association factor I have a decreased ability for polypeptide snythesis directed phage mRNA or poly(U).     

Isolation and characterization of cytoplasmic inclusions from influenza A virus-infected cells.

Influenza A viruses induce the accumulation of electron-dense inclusions in the cytoplasm of infected cells during the latter stages of the replication cycle. Cell fractionation studies showed that these inclusions could be recovered in subcellular fractions containing ribosomes and polysomes. Isolation of these inclusions was accomplished by procedures involving RNase treatment of these fractions followed by repurification, or by fluorocarbon extraction and gradient centrifugation. Electron microscopy indicated that the isolated inclusions exhibited a major periodicity of approximately 8 nm with minor periodicities of approximately 4 nm. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the influenza virus coded nonstructural protein was the only protein component present in isolated inclusions.     

GABA agonists and neurotransmitters metabolizing enzymes in steroid-primed OVX rats

The effect of intraventricular (IVT) administration of GABAA receptor agonist muscimol and GABAB receptor agonist, baclofen was examined on the activity of acetylcholinesterase (AChE), monoamine oxidase (MAO) and Na+, K+-ATPase in discrete areas of brain from estrogen-progesterone primed ovariectomized rats. AChE enzyme activity was increased in two subcellular fractions (soluble and total particulate) studied, with statistically significant changes in cerebral hemispheres (CH), cerebellum (CB), thalamus (TH) and hypothalamus (HT), Na+, K+-ATPase enzyme activity was decreased in both these fractions. MAO activity increased significantly in CH, TH and HT. The presented results suggest a functional relationship between GABAergic (inhibitory), cholinergic and monoaminergic (excitatory) systems by affecting the rate of degradation of the excitatory neurotransmitters and Na+, K+-ATPase. (Mol Cell Biochem 167: 107-111, 1997)     

Translation and the cytoskeleton: a mechanism for targeted protein synthesis

This review describes the critical evidence that in eukaryotic cells polyribosomes, mRNAs and components of the protein synthetic machinery are associated with the cytoskeleton. The role of microtubules, intermediate filaments and microfilaments are discussed; at present most evidence suggests that polyribosomes interact with the actin filaments. The use of non-ionic detergent/deoxycholate treatment in the isolation of cytoskeletal-bound polysomes is described and the conclusion reached that at low salt concentrations this leads to mixed preparations of polysomes derived from both the cytoskeleton and the endoplasmic reticulum. At present the best approach for isolation of cytoskeletal-bound polysomes appears to involve extraction with salt concentrations greater than 130 mM after an initial non-ionic detergent treatment. Such polysomes appear to be enriched in certain mRNAs and thus it is suggested that they are involved in translation of a unique set of proteins. The evidence for mRNA localisation is presented and the role of the cytoskeleton in transport and localisation of RNA discussed. Recent data on the role of the 3 untranslated region in the targeting of mRNAs both to particular regions of the cell and for translation on cytoskeletal-bound polysomes is described. The hypothesis is developed that the association of polysomes with the cytoskeleton is the basis of a mechanism for the targeting of mRNAs and the compartmentalization of protein synthesis.Abbreviations CBP cytoskeletal-bound polysomes - FP free polysomes - MBP membrane-bound polysomes - ER endoplasmic reticulum     

Cytoplasmic distribution of heat shock proteins in soybean

Previous analyses of the distribution of heat shock (hs) proteins in soybean (Glycine max L. Merr., var Wayne) have demonstrated that a fraction of the low molecular weight hs protein associates with ribosomes during hs. To more specifically characterize the nature of this association, isokinetic centrifugation of ribosomes through sucrose gradients was used to separate monosomes from polysomes. The present analysis demonstrated that hs proteins were bound to polysomes but not monosomes. Treatment of polysomes with puromycin, K⁺, and Mg²⁺, which caused dissociation of ribosomes into 40S and 60S subunits, also caused dissociation of the hs proteins. Using the procedure of Nover et al. (1983, Mol. Cell Biol, 3: 1628-1655), a hs granule fraction was also isolated. As in tomato cells, hs granules from soybean seedlings contained the low molecular weight hs proteins as a primary component and a number of other non-hs proteins of relative molecular mass 30 to 40 kilodaltons and 70 to 90 kilodaltons. On metrizamide gradients they exhibited a buoyant density of 1.20 to 1.21 grams per cubic centimeter, typical of ribonucleoprotein particles. Heat shock granules were characterized as unique cytoplasmic particles based on protein composition and buoyant density. Isopycnic centrifugation of ribosome preparations demonstrated that they contained hs granules, but the hs proteins bound to polysomes were not released by KCI/EDTA treatment. Thus, the polysome-bound hs proteins and the granule-bound hs proteins appear to represent two distinct populations of hs proteins in the cytoplasm. Heat shock granules were not distinguishable from ribosomes at the level of resolution used in transmission electron microscopy.     

The influence of temperature upon polysomes of spermatids of rat testes

Incorporation of [³H]phenylalanine into protein by a reconstituted lysate subcellular system (ribosomes plus high-speed supernatant) from rat spermatids was measured at 34°C after 5 minutes preincubation of one component at 0°C while the other component was incubated at temperatures from 30°C to 40°C. Preincubation at temperatures above 34°C inhibits the ribosomal activity but not the high-speed supernatant activity. The incubation of lysate above 34°C results from a dissociation of polysomes to monosomes. These results indicate that ribosomes are the most sensitive component to the increased temperature on protein synthesis in lysate cell free system by spermatids and that the inhibition of protein synthesis in spermatids above 34°C is at least partly explained by the breakdown of polysomes in these cells.     

Formation and stability of polysomes and polysomal populations in roots of germinating seeds of soybean (Glycine max L.) under cold,osmotic and combined cold and osmotic stress conditions

Abiotic stress factors such as extreme temperatures or osmotic stress are among the major causes of inferior crop yields. In response to a stress, plants have evolved various defense mechanisms. In our study, we have demonstrated how cold stress, osmotic stress and a combination of both stresses retard the growth of roots and inhibit the process of ribosomes binding into polysomes. The tested stresses also limited the ability of root tissues to synthesize proteins. At the same time, most of the analyzed samples were found to contain elevated shares of the fractions of cytoskeleton-bound polysomes (CBP, CMBP) in the total population of polysomes. Using a polysome-based degradation system, it was shown that polysomes formed under stress conditions were much more resistant to the effect of exogenous ribonuclease than the control ones. The highest tolerance to digestion was demonstrated by the cytoskeleton-bound (CBP) and cytoskeleton-membrane bound polysomes (CMBP). The increasing share of CBP and their stability in roots of seeds germinating under stress conditions can be a target for physiological regulation. It seems that modifications in the stability and percentages of particular polysomal populations play an important role in the adaptation of plants to stress conditions, which may indicate that these forms of polysomes, i.e., cytoskeleton-bound ones, are involved (via selective translation) in the synthesis of stress proteins in soybean roots.     

Translational control of protein synthesis after infection by vesicular stomatitis virus.

Four hours after infection of BHK cells by vesicular stomatitis virus (VSV), the rate of total protein synthesis was about 65% that of uninfected cells and synthesis of the 12 to 15 predominant cellular polypeptides was reduced to a level about 25% that of control cells. As determined by in vitro translation of isolated RNA and both one- and two-dimensional gel analyses of the products, all predominant cellular mRNA's remained intact and translatable after infection. The total amount of translatable mRNA per cell increased about threefold after infection; this additional mRNA directed synthesis of the five VSV structural proteins. To determine the subcellular localization of cellular and viral mRNA before and after infection, RNA from various sizes of polysomes and nonpolysomal ribonucleoproteins (RNPs) was isolated from infected and noninfected cells and translated in vitro. Over 80% of most predominant species of cellular mRNA was bound to polysomes in control cells, and over 60% was bound in infected cells. Only 2 of the 12 predominant species of translatable cellular mRNA's were localized to the RNP fraction, both in infected and in uninfected cells. The average size of polysomes translating individual cellular mRNA's was reduced about two- to threefold after infection. For example, in uninfected cells, actin (molecular weight 42,000) mRNA was found predominantly on polysomes with 12 ribosomes; after infection it was found on polysomes with five ribosomes, the same size of polysomes that were translating VSV N (molecular weight 52,000) and M (molecular weight 35,000) mRNA. We conclude that the inhibition of cellular protein synthesis after VSV infection is due, in large measure, to competition for ribosomes by a large excess of viral mRNA. The efficiency of initiation of translation on cellular and viral mRNA's is about the same in infected cells; cellular ribosomes are simply distributed among more mRNA's than are present in growing cells. About 20 to 30% of each of the predominant cellular and viral mRNA's were present in RNP particles in infected cells and were presumably inactive in protein synthesis. There was no preferential sequestration of cellular or viral mRNA's in RNPs after infection.     

Co-localization of polysomes,cytoskeleton, and membranes with protein bodies from corn endosperm

Summary Frozen corn endosperm was homogenized in a cytoskeleton-stabilizing buffer and stained directly (without pelleting) with rhodamine-phalloidin for actin and either thiazole orange to stain RNA or DiOC₆ to stain membranes prior to examination under the fluorescence microscope. Other samples were treated with a non-ionic detergent alone or in conjunction with a ionic detergent prior to staining and fluorescence microscopy. Very gentle homogenization in unsupplemented buffer yielded a massive aggregate containing protein bodies that fluoresced after treatment with the ER stain DiOC₆. This aggregate was capped by an aggregate of unstained starch grains. More vigorous homogenization yielded more disperse patterns showing almost identical co-localization of ER, actin and RNA (polysomes). Homogenization in buffer plus non-ionic detergent removed most of the membrane yet maintained co-localization of actin and polysomes, while homogenization in double detergent removed the last traces of membrane and actin, and released over 70% of the polysomes. We interpret these results to suggest that protein bodies are surrounded by membranes, cytoskeleton and RNA (polysomes) and that the majority of the polysomes are attached more firmly to the cytoskeleton than to the membrane. This provides evidence from fluorescence microscopy to supplement that from biochemical analyses for the existence of cytomatrix-bound polysomes in plants.Abbreviations CBP cytoskeleton-bound polysomes - CMBP cyto-matrix-bound polysomes - CSB cytoskeleton-stabilizing buffer - DOC sodium deoxycholate - DiOC₆ 3,3-dihexyloxacarbocyanine iodide - DTE dithioerythritol - MBP membrane-bound polysomes - FP free polysomes - PMSF phenylmethyl-sulfonyl fluoride - PTE polyoxy-ethylene-10-tridecyl ether - Rh-Ph rhodamine-phalloidin - TO thiazole orange - Tris tris-(hydroxymethyl) aminomethane     

Biosynthesis of microsomal nicotinamide–adenine dinucleotide phosphate–cytochrome c reductase by membrane-bound and free polysomes from rat liver

1. NADPH-ferricytochrome c oxidoreductase (EC 1.6.2.3) was purified from the endoplasmic reticulum of rat liver cells. The methods, which involved digestion of membrane with Steapsin, a crude pancreatic extract containing diastase and trypsin, gel filtration and preparative electrophoresis on polyacrylamide, provided an enzyme with a high specific activity in good yield. 2. The incorporation of (14)C-labelled amino acids into the purified reductase by the incubation of various subcellular fractions was studied. The microsome fraction, bound polysomes, free polysomes and detergent-treated polysomes effected the synthesis of the enzyme. 3. The reductase that had been synthesized by the polysomes was tightly bound to preparations of smooth-surfaced endoplasmic reticulum that were added to the incubation medium. 4. Reductase activity could be detected on both free and detergent-treated polysomes. Evidence is presented to show that this activity was due, at least in part, to the presence on the ribosomes of nascent enzyme. The association of enzyme with detergent-treated polysomes did not appear to be due to contamination of the ribosomes with either membrane or cell sap but it is possible for such ribosomes to adsorb some enzyme. 5. The amount of reductase activity associated with the detergent-treated polysomes was increased when the rats from which the polysomes were derived had been previously injected with phenobarbitone. 6. The results are discussed with respect to their relevance for the question of the existence of two functionally different groups of polysomes in the liver and for current ideas on the biogenesis of membranes.