scholarly journals All factors required for protein synthesis are retained on heparin bound to Sepharose

1978 ◽  
Vol 172 (1) ◽  
pp. 1-7 ◽  
Author(s):  
J Hradec ◽  
Z Dušek
Keyword(s):  
Protein Synthesis ◽  
Affinity Chromatography ◽  
Rat Liver ◽  
Mammalian Cells ◽  
Gel Filtration ◽  
Ribosomal Subunits ◽  
Messenger Ribonucleoprotein ◽  
Reticulocyte Lysates ◽  
Sepharose 4B

1. Postmitochondrial supernatants of rabbit reticulocyte lysates were chromatographed on heparin bound to Sepharose 4B, and the fraction retained on affinity columns was separated by subsequent gel filtration on Sepharose 4B into three fractions, two of them active in protein synthesis. 2. The heavier fraction sedimented at 40S and contained more than 10% RNA. This consisted predominantly of a 12S component, with smaller amounts of the 9S and 4S RNA species. The lighter fraction (18-20S) was composed of proteins with less than 1% RNA. 3. Different enzymic activities were associated with these fractions. 4. In the presence of both fractions, efficient translation took place on combined ribosomal subunits of rat liver with added cofactors. Globin messenger ribonucleoprotein stimulated this translation 5-6-fold. 5. Relatively large complexes of all factors required for protein synthesis are apparently isolated from reticulocytes by affinity chromatography on heparin-Sepharose 4B. Such complexes may occur naturally in the cytoplasm of mammalian cells.

Purification Of Vasoactive Peptides From Human Fibrinogen Degraded By Human Leucocyte Elastase

1981 ◽  
Author(s):  
R Wallin ◽  
M Belew ◽  
K Ohlsson ◽  
T Saldeen
Keyword(s):  
Affinity Chromatography ◽  
Vascular Permeability ◽  
Gel Filtration ◽  
Molar Ratio ◽  
Significant Activity ◽  
Small Peptides ◽  
Human Fibrinogen ◽  
Leucocyte Elastase ◽  
Pm 10 ◽  
Sepharose 4B

The presence of leucocytes around extravascular fibrin deposits suggests that the leucocyte elastases might be partly responsible for the extravascular degradation of fibrin. Our previous studies have shown that the degradation of fibrin(ogen) by plasmin leads to the release of 2 small peptides which markedly increase vascular permeability and induce oedema e.g. in the lungs. The results of this investigation show that small peptides released from fibrinogen after degradation by leucocytes elastases also increase vascular permeability.Human fibrinogen (Kabi, Grade L) was made plasminogenfree by affinity chromatography on Lysine-Sepharose 4B prior to use. The human leucocyte elastases were isolated from extracts of lysosome-like granules of human leukaemic myeloid cells by a combination of gel filtration, affinity chromatography and preparative agarose gel electrophoresis. The fibrinogen (0.5 %) and the leucocyte elastases (in a molar ratio of 100:1) were incubated together for 48 h at +37°C and at pH 8.5. The mixture was then cooled to +4°C to stop the lysis and ultrafiltrated on a DIAFLO PM 10 membrane until the retentate was approximately 10 % of the starting volume. The peptides in the diffusate accounted for about 20 % of the starting material as estimated from absorbance measurements at 280 nm. The diffusate was concentrated by lyophilization and fractionated by chromatography on a column of Bio-Gel P-6. At least 8 fractions were obtained of which only two showed a significant activity in their ability to increase vascular permeability in rat skin. The active peptides in these two fractions were further purified to homogeneity by column zone electrophoresis at various pHs and their amino acid compositions established.

scholarly journals Nuclease activity associated with mammalian mRNA in its native state: possible basis for selectivity in mRNA decay.

1990 ◽  
Vol 10 (5) ◽  
pp. 2060-2069 ◽  
Author(s):  
R Bandyopadhyay ◽  
M Coutts ◽  
A Krowczynska ◽  
G Brawerman
Keyword(s):  
Mammalian Cells ◽  
Nuclease Activity ◽  
Noncoding Region ◽  
Beta Globin ◽  
Native State ◽  
Globin Mrna ◽  
Ribosomal Subunits ◽  
Messenger Ribonucleoprotein ◽  
Mouse Sarcoma ◽  
Actin Mrna

Polysome and messenger ribonucleoprotein (mRNP) preparations from various mammalian cells contain tightly bound nuclease activity that causes degradation of the mRNA in the preparations. This activity was found to cosediment with all polysome size classes as well as with free mRNPs and to remain associated with the mRNPs released from polysomes by treatment with EDTA. No association with ribosomal subunits was evident. The rates of mRNA degradation were not affected by serial dilution, an indication that enzyme and substrate are tightly associated. beta-Globin mRNA in purified reticulocyte polysomes was cleaved at AU sequences in the 3'-terminal region. Cleavages at the same sites occurred when deproteinized reticulocyte RNA was incubated with mouse sarcoma 180 (S-180) polysomes. The S-180 preparations caused additional cleavages, primarily at UG sequences. A P40 mRNA in S-180 polysomes was cleaved primarily in the 3' noncoding region, but the cleavages in a P21 mRNA were seen in the 5' noncoding region only. Actin mRNA was cleaved in an internal region, yielding large relatively stable 3'- and 5'-terminal fragments. These data suggest the occurrence of highly specific interactions between one or more mRNA-bound nucleases and individual mRNA species.

scholarly journals Identification of rat liver phosphatidylinositol synthase as a 21 kDa protein

1994 ◽  
Vol 304 (1) ◽  
pp. 301-305 ◽  
Author(s):  
M E Monaco ◽  
M Feldman ◽  
D L Kleinberg
Keyword(s):  
Heat Treatment ◽  
Enzyme Activity ◽  
Affinity Chromatography ◽  
Molecular Mass ◽  
Rat Liver ◽  
Protein Band ◽  
Absolute Requirement ◽  
Sds Page ◽  
Sepharose 4B ◽  
Post Mitochondrial Supernatant

Substantial purification of rat liver phosphatidylinositol (PtdIns) synthase has been achieved by a combination of Hecameg extraction, heat treatment, affinity chromatography and chromatography on PBE-94. The activity chromatographs as a single peak which has an apparent molecular mass between 150 and 200 kDa on Sepharose 4B. When analysed by SDS/PAGE, two major bands are seen. The enzyme activity is correlated with a protein band of 21 kDa. A second band, at 51 kDa, is eluted from a PBE-94 column slightly ahead of the activity. Manganese is an absolute requirement for stabilization of activity in the presence of detergent. The effect of manganese is optimal at 0.5 mM; magnesium at a concentration of 10 mM is only minimally effective. Substrate Kms are 1.3 mM and 9.5 microM for inositol and CDP-diacylglycerol respectively. The activity eluting from the PBE-94 column is purified 5000-fold over the post-mitochondrial supernatant.

scholarly journals Characterization of a rat liver factor that inhibits initiation of protein synthesis in rabbit reticulocyte lysates

1977 ◽  
Vol 74 (6) ◽  
pp. 2264-2268 ◽  
Author(s):  
J. Delaunay ◽  
R. S. Ranu ◽  
D. H. Levin ◽  
V. Ernst ◽  
I. M. London
Keyword(s):  
Protein Synthesis ◽  
Rat Liver ◽  
Reticulocyte Lysates

scholarly journals eIF6 rebinding dynamically couples ribosome maturation and translation

2021 ◽  
Author(s):  
Pekka Jaako ◽  
Alexandre Faille ◽  
Shengjiang Tan ◽  
Chi C Wong ◽  
Norberto Escudero-Urquijo ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Translation Initiation ◽  
Mammalian Cells ◽  
Ribosomal Subunits ◽  
Inactive State ◽  
Global Protein Synthesis ◽  
Cyclical Process ◽  
Shwachman Diamond Syndrome ◽  
Subunit Joining

Protein synthesis is a cyclical process consisting of translation initiation, elongation, termination and ribosome recycling. The release factors SBDS and EFL1 (both mutated in the leukaemia predisposition disorder Shwachman-Diamond syndrome) license entry of nascent 60S ribosomal subunits into active translation by evicting the anti-association factor eIF6 from the 60S intersubunit face. Here, we show that in mammalian cells, eIF6 holds all free cytoplasmic 60S subunits in a translationally inactive state and that SBDS and EFL1 are the minimal components required to recycle these 60S subunits back into additional rounds of translation by evicting eIF6. Increasing the dose of eIF6 in mice in vivo impairs terminal erythropoiesis by sequestering post-termination 60S subunits in the cytoplasm, disrupting subunit joining and attenuating global protein synthesis. Our data reveal that ribosome maturation and recycling are dynamically coupled by a mechanism that is disrupted in an inherited leukaemia predisposition disorder.

Nuclease activity associated with mammalian mRNA in its native state: possible basis for selectivity in mRNA decay

1990 ◽  
Vol 10 (5) ◽  
pp. 2060-2069
Author(s):  
R Bandyopadhyay ◽  
M Coutts ◽  
A Krowczynska ◽  
G Brawerman
Keyword(s):  
Mammalian Cells ◽  
Nuclease Activity ◽  
Noncoding Region ◽  
Beta Globin ◽  
Native State ◽  
Globin Mrna ◽  
Ribosomal Subunits ◽  
Messenger Ribonucleoprotein ◽  
Mouse Sarcoma ◽  
Actin Mrna

Polysome and messenger ribonucleoprotein (mRNP) preparations from various mammalian cells contain tightly bound nuclease activity that causes degradation of the mRNA in the preparations. This activity was found to cosediment with all polysome size classes as well as with free mRNPs and to remain associated with the mRNPs released from polysomes by treatment with EDTA. No association with ribosomal subunits was evident. The rates of mRNA degradation were not affected by serial dilution, an indication that enzyme and substrate are tightly associated. beta-Globin mRNA in purified reticulocyte polysomes was cleaved at AU sequences in the 3'-terminal region. Cleavages at the same sites occurred when deproteinized reticulocyte RNA was incubated with mouse sarcoma 180 (S-180) polysomes. The S-180 preparations caused additional cleavages, primarily at UG sequences. A P40 mRNA in S-180 polysomes was cleaved primarily in the 3' noncoding region, but the cleavages in a P21 mRNA were seen in the 5' noncoding region only. Actin mRNA was cleaved in an internal region, yielding large relatively stable 3'- and 5'-terminal fragments. These data suggest the occurrence of highly specific interactions between one or more mRNA-bound nucleases and individual mRNA species.

scholarly journals Messenger Ribonucleoprotein Particles from Plant Cell Cultures

1978 ◽  
Vol 33 (5-6) ◽  
pp. 359-362 ◽  
Author(s):  
Jörg Pfisterer
Keyword(s):  
Affinity Chromatography ◽  
Size Distribution ◽  
Plant Cell ◽  
Cell Cultures ◽  
Buoyant Density ◽  
Plant Cell Cultures ◽  
Ribosomal Subunits ◽  
Ribonucleoprotein Particles ◽  
Messenger Ribonucleoprotein ◽  
Labeled Rna

Abstract Messenger ribonucleoprotein particles were isolated from the polysomes of logarithmically growing plant cell cultures, pulse-labeled with [3H] adenosine for 30 min. More than 80% of the labeled RNP was present in particles sedimenting between 80 S and 30 S on sucrose density gradients, but was not associated with ribosomal subunits. The size distribution differs from those reported for polysomal mRNP particles to date. After fixation with glutaraldehyde the labeled RNP particles had a buoyant density of 1.38 g/cm3 in CsCl gradients. Radioactively labeled RNA extracted from the RNP particles showed a heterodisperse size distribution and contained poly (A) stretches as determined by affinity chromatography and ribonuclease digestion experiments.

scholarly journals Purification and properties of different forms of modeccin, the toxin of Adenia digitata. Separation of subunits with inhibitory and lectin activity

1980 ◽  
Vol 185 (1) ◽  
pp. 203-210 ◽  
Author(s):  
L Barbieri ◽  
M Zamboni ◽  
L Montanaro ◽  
S Sperti ◽  
F Stirpe
Keyword(s):  
Protein Synthesis ◽  
Affinity Chromatography ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Polyacrylamide Gel ◽  
Purification And Properties ◽  
Inhibitor Of Protein Synthesis ◽  
Sepharose 4B

1. The subunits were isolated of modeccin (subsequently referred to as modeccin 4B), the toxin purified from the roots of Adenia digitata by affinity chromatography on Sepharose 4B [Gasperi-Campani, Barbieri, Lorenzoni, Montanaro, Sperti, Bonetti & Stirpe (1978) Biochem J. 174, 491-496]. They are an A subunit (mol.wt. 26 000), which inhibits protein synthesis, and a B subunit (mol.wt. 31 000), which binds to cells. Both sununits, as well as intact modeccin, gave single bands on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, but showed some heterogeneity on isoelectric focusing and on polyacrylamide-gel electrophoresis at pH 9.5. 2. A second form of modeccin, not retained by Sepharose 4B, was purified by affinity chromatography on acid-treated Sepharose 6B: this form is subsequently termed modeccin 6B 3. Modeccin 6B has a molecular weight indistinguishable from that of modeccin 4B, and consists of two subunits of mol.wts. 27 000 and 31 000, joined by a disulphide bond. The subunits were not isolated because of their high insolubility in the absence of sodium dodecyl sulphate. 4. As compared with modeccin 4B, modeccin 6B is slightly less toxic to animals, does not agglutinate erythrocytes, and is a more potent inhibitor of protein synthesis in a lysate of rabbit reticulocytes, giving 50% inhibition at the concentration of 0.31 microgram/ml.

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