scholarly journals RIBOSOME-MEMBRANE INTERACTION

1973 ◽  
Vol 56 (1) ◽  
pp. 206-229 ◽  
Author(s):  
M. R. Adelman ◽  
David D. Sabatini ◽  
Günter Blobel
Keyword(s):  
Ionic Strength ◽  
Room Temperature ◽  
Ribosomal Subunit ◽  
High Ionic Strength ◽  
Ribosomal Subunits ◽  
Insoluble Material ◽  
Polyuridylic Acid ◽  
Nascent Chain ◽  
Membrane Bound ◽  
Polypeptide Chains

In a medium of high ionic strength, rat liver rough microsomes can be nondestructively disassembled into ribosomes and stripped membranes if nascent polypeptides are discharged from the bound ribosomes by reaction with puromycin. At 750 mM KCl, 5 mM MgCl2, 50 mM Tris·HCl, pH 7 5, up to 85% of all bound ribosomes are released from the membranes after incubation at room temperature with 1 mM puromycin. The ribosomes are released as subunits which are active in peptide synthesis if programmed with polyuridylic acid. The ribosome-denuded, or stripped, rough microsomes (RM) can be recovered as intact, essentially unaltered membranous vesicles Judging from the incorporation of [3H]puromycin into hot acid-insoluble material and from the release of [3H]leucine-labeled nascent polypeptide chains from bound ribosomes, puromycin coupling occurs almost as well at low (25–100 mM) as at high (500–1000 mM) KCl concentrations. Since puromycin-dependent ribosome release only occurs at high ionic strength, it appears that ribosomes are bound to membranes via two types of interactions: a direct one between the membrane and the large ribosomal subunit (labile at high KCl concentration) and an indirect one in which the nascent chain anchors the ribosome to the membrane (puromycin labile). The nascent chains of ribosomes specifically released by puromycin remain tightly associated with the stripped membranes. Some membrane-bound ribosomes (up to 40%) can be nondestructively released in high ionic strength media without puromycin; these appear to consist of a mixture of inactive ribosomes and ribosomes containing relatively short nascent chains. A fraction (∼15%) of the bound ribosomes can only be released from membranes by exposure of RM to ionic conditions which cause extensive unfolding of ribosomal subunits, the nature and significance of these ribosomes is not clear.

Functional ramifications of FRET-detected nascent chain folding far inside the membrane-bound ribosome

2004 ◽  
Vol 32 (5) ◽  
pp. 668-672 ◽  
Author(s):  
A.E. Johnson
Keyword(s):  
Protein Trafficking ◽  
Protein Biosynthesis ◽  
Ribosomal Subunit ◽  
Regulatory Control ◽  
Secretory Proteins ◽  
Induced Folding ◽  
Ribosomal Tunnel ◽  
Nascent Chain ◽  
Membrane Bound ◽  
Transmembrane Sequence

During protein biosynthesis, nascent protein chains are directed along a long narrow tunnel that spans the large ribosomal subunit. It has recently become clear that this structural feature has evolved to effect regulatory control over aspects of protein synthesis and protein trafficking. Since this control is nascent chain-specific, ribosomal components that form the tunnel must be involved in recognizing selected nascent proteins as they pass by. The present study focuses on one such situation in which nascent secretory proteins and membrane proteins are distinguished by the ribosome-induced folding of the latter's hydrophobic transmembrane sequence far inside the ribosomal tunnel and close to the peptidyltransferase centre.

scholarly journals Experiments and Thermodynamic Modeling of Chukanovite (Fe2(OH)2CO3) to High Ionic Strengths

MRS Advances ◽  
2017 ◽  
Vol 2 (13) ◽  
pp. 747-752 ◽  
Author(s):  
Sungtae Kim ◽  
Justin Dean ◽  
Jandi Knox ◽  
Leslie Kirkes ◽  
Je-Hun Jang
Keyword(s):  
Free Energy ◽  
Ionic Strength ◽  
Room Temperature ◽  
Xrd Analysis ◽  
High Ionic Strength ◽  
Model Parameters ◽  
Second Phase ◽  
Experimental Conditions ◽  
Ph Values ◽  
Decreasing Ph

ABSTRACTWhile conducting siderite (FeCO3) solubility experiments in NaCl-Na2CO3 brines, evidence for a second phase was detected. Experiments, in which synthesized siderite was reacted with high ionic strength (0.18 – 7.5 m) solutions at room temperature and high pH (>10), were conducted in a glovebox. As the aging time of siderite-bearing experiments increased, the pH of the solution decreased, signaling formation of a hydroxyl-bearing phase. Decreasing pH values are interpreted to indicate that a hydroxyl-bearing phase, such as chukanovite, is the reaction controlling solid in the solid assemblage. Chukanovite was tentatively identified by XRD analysis. We set out, therefore, to determine the thermodynamic stability of chukanovite under the experimental conditions. Aqueous thermodynamic model parameters were determined with experimentally analyzed Fe(II) solubility data, and subsequently yielded a proposed formation free energy of chukanovite (-1149.8 kJ/mol).

scholarly journals Retention of mRNA on the endoplasmic reticulum membranes after in vivo disassembly of polysomes by an inhibitor of initiation.

1976 ◽  
Vol 71 (1) ◽  
pp. 307-313 ◽  
Author(s):  
M Adesnik ◽  
M Lande ◽  
T Martin ◽  
D D Sabatini
Keyword(s):  
Messenger Rna ◽  
Membrane Fraction ◽  
Human Fibroblasts ◽  
Ribosomal Subunits ◽  
Run Off ◽  
Microsomal Membranes ◽  
Membrane Bound ◽  
Polypeptide Chains ◽  
Extensive Release

Membrane-bound ribosomes and messenger RNA remained associated with the microsomal membranes of human fibroblasts after cultures were treated with Verrucarin A, an inhibitor of initiation which led to extensive run-off of ribosomes from polysomal structures. When a membrane fraction from Verrucarin-treated cells containing such inactive ribosomes and mRNA was suspended in a medium of high salt concentration, extensive release of ribosomal subunits occurred without the need for puromycin. The mRNA nevertheless remained associated with the membranes. These results add support to the conclusion that, in human fibroblasts, mRNA is bound directly to ER membranes, independently of the ribosomes and nascent polypeptide chains.

scholarly journals RIBOSOME CRYSTALLIZATION IN CHICKEN EMBRYOS

1972 ◽  
Vol 52 (2) ◽  
pp. 338-354 ◽  
Author(s):  
T. Morimoto ◽  
G. Blobel ◽  
D. D. Sabatini
Keyword(s):  
Messenger Rna ◽  
Ribosomal Proteins ◽  
Ribosomal Subunits ◽  
Chicken Embryos ◽  
Polyuridylic Acid ◽  
Insoluble Product ◽  
Rnase Treatment ◽  
Low Ionic Strength ◽  
Polypeptide Chains

Isolated tetrameric particles (166S) derived from the crystalline lattices known to appear in hypothermic chicken embryos consist of mature 80S ribosomes which contain all species of ribosomal RNA and a complete set of ribosomal proteins. Ribosome tetramers are not a special type of polysomes since in solutions of high ionic strengths (500 mM KCl and 50 nM triethanolamine-HCl buffer) containing 5 mM MgCl2 they dissociate into 40S and 60S ribosomal subunits, without the need of puromycin, and at a concentration of Mg++ higher than 3 mM they are not disassembled by mild RNase treatment. Tetramers spontaneously disassemble into 80S monomers when the Mg++ concentration is lowered to 1 mM at relatively low ionic strength. Tetramers failed to couple in vitro puromycin-3H into an acid-insoluble product, indicating the lack of nascent polypeptide chains. Although tetramers have no endogenous messenger RNA activity, they can be programmed in vitro with polyuridylic acid (poly U) to synthesize polyphenylalanine. All ribosomes within a tetramer can accept poly U, without the need of disassembly of the tetramers into monomers or subunits.

scholarly journals The effects of polyuridylic acid on phenylalanine incorporation by subcellular fractions from carbon tetrachloride-poisoned rat liver

1968 ◽  
Vol 107 (2) ◽  
pp. 151-163 ◽  
Author(s):  
Edward A. Smuckler ◽  
Benno Parthier ◽  
Tore Hultin
Keyword(s):  
Amino Acid ◽  
Ionic Strength ◽  
Carbon Tetrachloride ◽  
Specific Activity ◽  
High Ionic Strength ◽  
Carbon Tetrachloride Poisoning ◽  
Optimum Range ◽  
Polyuridylic Acid ◽  
Isolated Structures ◽  
Endogenous Activity

1. Ribosomes and microsomes isolated from the livers of rats that had received carbon tetrachloride 1hr. previously had decreased endogenous capacity to incorporate amino acid. 2. The capacity of the isolated structures to respond to a synthetic messenger, polyuridylic acid, and to incorporate phenylalanine was investigated. 3. It was found that ribosomes from carbon tetrachloride-treated animals, prepared with detergent and at high ionic strength, could be restored to the same specific activity as control particles with polyuridylic acid but that these particles required more Mg2+ in the incubation mixture. 4. Microsomes could also be stimulated to control activities with polyuridylic acid, but had a narrow optimum range of Mg2+ concentration. 5. Microsomes prepared from poisoned animals could be preprogrammed with polyuridylic acid to a significantly greater degree than could control particles, and this response was greater with increasing Mg2+ concentrations. These data suggested that in carbon tetrachloride poisoning the messenger–ribosome interaction had been altered. 6. Attempts to deprogramme particles from control and treated animals resulted in decreased endogenous activity of both particles and a decreased capacity for the treated particles to be restored with the synthetic messenger. 7. It is suggested that two effects are present in carbon tetrachloride poisoning, namely an alteration of the messenger–ribosome interaction and an increased lability of the ribosome, as either separate or related events.

Three-dimensional reconstruction of the 40S ribosomal subunit from rabbit reticulocytes

1987 ◽  
Vol 45 ◽  
pp. 936-937
Author(s):  
Neng-Yu Zhang ◽  
Terence Wagenknecht ◽  
Michael Radermacher ◽  
Tom Obrig ◽  
Joachim Frank
Keyword(s):  
Three Dimensional ◽  
Ribosomal Subunit ◽  
Uranyl Acetate ◽  
Reconstruction Method ◽  
Single Exposure ◽  
Electron Dose ◽  
Ribosomal Subunits ◽  
40S Ribosomal Subunit ◽  
Dimensional Reconstruction ◽  
Rabbit Reticulocytes

We have reconstructed the 40S ribosomal subunit at a resolution of 4 nm using the single-exposure pseudo-conical reconstruction method of Radermacher et al.Small (40S) ribosomal subunits were Isolated from rabbit reticulocytes, applied to grids and negatively stained (0.5% uranyl acetate) in a manner that “sandwiches” the specimen between two layers of carbon. Regions of the grid exhibiting uniform and thick staining were identified and photographed twice (magnification 49,000X). The first micrograph was always taken with the specimen tilted by 50° and the second was of the Identical area untilted (Fig. 1). For each of the micrographs the specimen was subjected to an electron dose of 2000-3000 el/nm2.Three hundred thirty particles appearing in the L view (defined in [4]) were selected from both tilted- and untilted-specimen micrographs. The untilted particles were aligned and their rotational alignment produced the azimuthal angles of the tilted particles in the conical tilt series.

Importance of Protease Inhibition in Studies on Purified Factor VIII (Antihaemophilic Factor)

1976 ◽  
Vol 35 (01) ◽  
pp. 186-190 ◽  
Author(s):  
Eugen A. Beck ◽  
Peter Bachmann ◽  
Peter Barbier ◽  
Miha Furlan
Keyword(s):  
Ionic Strength ◽  
Factor Viii ◽  
Gel Filtration ◽  
High Ionic Strength ◽  
Procoagulant Activity ◽  
Carrier Protein ◽  
Protease Inhibition ◽  
Functional Sites ◽  
Molecular Weight Peak ◽  
Von Willebrand

SummaryAccording to some authors factor VIII procoagulant activity may be dissociable from carrier protein (MW~ 2 × 106) by agarose gel filtration, e.g. at high ionic strength. We were able to reproduce this phenomenon. However, addition of protease inhibitor (Trasylol) prevented the appearance of low molecular weight peak of factor VIII procoagulant activity both at high ionic strength and elevated temperature (37°C). We conclude from our results that procoagulant activity and carrier protein (von Willebrand factor, factor VIII antigen) are closely associated functional sites of native factor VIII macro molecule. Consequently, proteolytic degradation should be avoided in functional and structural studies on factor VIII and especially in preparing factor VIII concentrate for therapeutic use.

THE RELATIVE DISTRIBUTION OF THYROXINE, TRIIODOTHYRONINE AND 3,3′,5′-(REVERSE)-TRIIODOTHYRONINE IN VARIOUS FRACTIONS OF THYROGLOBULIN

1978 ◽  
Vol 88 (2) ◽  
pp. 298-305 ◽  
Author(s):  
Peter Laurberg
Keyword(s):  
Ionic Strength ◽  
Guinea Pig ◽  
Sucrose Gradient ◽  
Deae Cellulose ◽  
High Ionic Strength ◽  
Relative Distribution ◽  
Thyroid Extract ◽  
Reverse Triiodothyronine ◽  
Thyroid Secretion ◽  
Stable Iodine

ABSTRACT Thyroglobulin fractions rich and poor in new thyroglobulin were separated by means of DEAE-cellulose chromatography of dog thyroid extracts and by zonal ultracentrifugation in a sucrose gradient of guinea pig thyroid extract incubated at low temperature. The distribution of thyroxine, triiodothyronine and 3,3′,5′-(reverse)-triiodothyronine in hydrolysates of the different fractions was estimated by radioimmunoassays. Following DEAE-cellulose chromatography there was a small but statistically significant increase in the T4/T3 ratio in thyroglobulin fractions eluted at high ionic strength - that is fractions relatively rich in stable iodine but poor in fresh thyroglobulin. There were no differences in the T4/rT3 ratios between the different fractions. The ratios between iodothyronines were almost identical in the various thyroglobulin fractions following zonal ultracentrifugation in a sucrose gradient of cold treated guinea pig thyroid extract. These findings lend no support to the possibility that a relatively high content of triiodothyronines in freshly synthesized thyroglobulin modulates the thyroid secretion towards a preferential secretion of triiodothyronine and 3,3′,5′-(reverse)-triiodothyronine at the expense of the secretion of thyroxine.

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