scholarly journals Release of poly a(+) messenger RNA from rat liver rough microsomes upon disassembly of bound polysomes

1977 ◽  
Vol 74 (2) ◽  
pp. 414-427 ◽  
Author(s):  
J Kruppa ◽  
DD Sabatini
Keyword(s):  
Ionic Strength ◽  
Rat Liver ◽  
Messenger Rna ◽  
High Salt ◽  
Salt Medium ◽  
Ribosomal Subunits ◽  
Microsomal Membranes ◽  
Low Ionic Strength

Several procedures were used to disassemble rat liver rough microsomes (RM) into ribosomal subunits, mRNA, and ribosome-stripped membrane vesicles in order to examine the nature of the association between the mRNA of bound polysomes and the microsomal membranes. The fate of the mRNA molecules after ribosome release was determined by measuring the amount of pulse-labeled microsomal RNA in each fraction which was retained by oligo-dT cellulose or by measuring the poly A content by hybridization to radioactive poly U. It was found that ribosomal subunits and mRNA were simultaneously released from the microsomal membranes when the ribosomes were detached by: (a) treatment with puromycin in a high salt medium containing Mg++, (b) resuspension in a high salt medium lacking Mg++, and (c) chelation of Mg++ by EDTA or pyrophosphate. Poly A-containing mRNA fragments were extensively released from RM subjected to a mild treatment with pancreatic RNase in a medium of low ionic strength. This indicates that the 3' end of the mRNA is exposed on the outer microsomal surface and is not directly bound to the membranes. Poly A segments of bound mRNA were also accessible to [(3)H] poly U for in situ hybridization in glutaraldehyde-fixed RM. Rats were treated with drugs which inhibit translation after formation of the first peptide bonds or interfere with the initiation of protein synthesis. After these treatments inactive monomeric ribosomes, as well as ribosomes bearing mRNA, remained associated with their binding sites in microsomes prepared in media of low ionic strength. However, because there were no linkages provided by nascent chains, ribosomes, and mRNA, molecules were released from the microsomal membranes without the need of puromycin, by treatment with a high salt buffer containing Mg++. Thus, both in vivo and in vitro observations are consistent with a model in which mRNA does not contribute significantly to the maintenance of the interaction between bound polysomes and endoplasmic reticulum membranes in rat liver hepatocytes.

Preparation Of Antithrombin, High Activity Heparin And A Heparin-Antithrombin Complex By A Rapid Two-Step Affinity Method

1981 ◽  
Author(s):  
R Jordan ◽  
T Zuffi ◽  
M Fournel ◽  
D Schroeder
Keyword(s):  
Ionic Strength ◽  
High Activity ◽  
Tight Binding ◽  
Therapeutic Benefit ◽  
Purification Scheme ◽  
The Matrix ◽  
Low Ionic Strength ◽  
Potential Therapeutic Benefit

The tight binding affinity of antithrombin for heparin makes possible a relatively selective purification scheme based on salt elution from heparin-Sepharose. We have found, however, that purity can often be greatly increased if the elution is carried out with soluble heparin instead. This heparin can be removed from the antithrombin, either in whole or part, by a second affinity step on Concanavalin A Sepharose. The antithrombin, which binds to the matrix through its glycosidic moieties, retains its ability to bind heparin at physiological ionic strengths. Thus, the complex of antithrombin and heparin is readily isolated free of unbound heparin species. The complex can be eluted intact with low ionic strength buffers containing sugars which compete for binding to the lectin. Alternatively, the high activity heparin (400–500 units/mg) can be obtained separately by a 1 M NaCl wash which is then followed by a carbohydrate wash to obtain the purified antithrombin.We have made certain preliminary biochemical and anticoagulant characterizations of these materials. Not unexpectedly, both the high activity heparin and its complex with antithrombin show significantly greater in vitro potency in comparison to unfractionated heparin. In vivo anticoagulant efficacy, as evaluated in a rabbit infusion model, confirmed the in vitro findings and further suggests some potential therapeutic benefit may be derived from infusion of a preformed heparin-antithrombin complex.

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 Differences between newly formed and recycled free small ribosome subunits in liver cytoplasm

1976 ◽  
Vol 158 (1) ◽  
pp. 97-103 ◽  
Author(s):  
R H Hinton ◽  
B M Mullock
Keyword(s):  
Ionic Strength ◽  
Rat Liver ◽  
Ribosomal Subunits ◽  
Additional Protein ◽  
Low Ionic Strength

Heterogeneity among the free small ribosomal subunits of rat liver was studied. Newly made free small subunits differ from the bulk of the free subunits in the following ways. (1) The newly made free small subunits sediment slightly faster than most free small subunits and form a band at slightly lower densities. (2) Mature free small subunits, prepared under conditions which minimize the amount of loosely bound protein, readily dimerize in solutions of low ionic strength. Little dimerization is found with newly formed subunits. This permits the separation of fractions enriched in newly formed subunits. (3) The fraction enriched in newly formed subunits contains a distinctive additional protein as compared the most of the free small subunits.

scholarly journals Biogenesis of microsomal membrane glycoproteins in rat liver. II. Purification of soluble glycoproteins and their incorporation into microsomal membranes.

1975 ◽  
Vol 67 (3) ◽  
pp. 700-714 ◽  
Author(s):  
F Autuori ◽  
H Svensson ◽  
G Dallner
Keyword(s):  
Rat Liver ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Exchange Chromatography ◽  
Microsomal Membrane ◽  
Membrane Glycoproteins ◽  
In Vivo Labeling ◽  
Microsomal Membranes

Sialoproteins isolated from the soluble fraction of rat liver could be incorporated into microsomal membranes. This incorporation was dependent on protein concentration, time, and temperature. Sodium dodecyl sulfate gel electrophoresis of membrane proteins after in vitro incorporation showed four major sugar-containing peaks and was similar to that found after in vivo labeling. Most of the incorporated protein was tightly bound to the microsomal membrane. Gel filtration and ion-exchange chromatography revealed the presence of several cytosolic glycoproteins that could be incorporated into microsomes. During prolonged centrifugation in a KBr solution with a density of 1.21 a highly labeled ([3H]glucosamine) protein (mole wt approximately to 70,000) that was actively incorporated into microsomes could be recovered in the upper region of the tube. These results demonstrate that several cytoplasmic glycoproteins of rat liver are transferred into microsomal membranes and that one of these is a lipoprotein.

Effect of X-irradiation on gene expression of rat liver chemokines: in vivo and in vitro studies

2008 ◽  
Vol 46 (01) ◽  
Author(s):  
F Moriconi ◽  
H Christiansen ◽  
H Christiansen ◽  
N Sheikh ◽  
J Dudas ◽  
...  
Keyword(s):  
Gene Expression ◽  
Rat Liver ◽  
In Vitro Studies ◽  
X Irradiation
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