Characterization of the particulate and free proteins obtained after treatment of ribosomes with 2 m-lithium chloride (original) (raw)
Related papers
1972
Abbreviations. RNA * protein, ribonucleoprotein; sarkosyl, N-lauryl sarcosine. Enzymes. T, ribonuclease (EC 2.7.7.26); pancreatic ribonuclease (EC 2.7.7.16). Definition. An A,,, unit is the quantity of material contained in 1 ml of a solution which has an absorbance of 1 at 260 nm, when measured in a 1-em path length cell. tions. After grinding with alumina [a], the cell paste was extracted with 10 mM MgC1, rather than 0.1 mM, to maintain the ribosomes as 70-5 particles. These 70-5 particles were washed by spinning through 0.5 M NH4Cl, 10 mM MgCl,, 10 mM Tris-HC1 pH 7.6 (cf. [5]), and were then dissociated into subparticles by resuspending in 50mM KC1, 0.3 mM MgCl,, 10mM Tris-HC1 p H 7.6. The subparticles were separated in a zonal rotor as before [I], and the 30-5 ribosomes precipitated with ethanol. The precipitate was dissolved in 0.3 mM magnesium acetate, 10 mM Tris-HC1 pH 7.6, and dialysed against this buffer, or against 1 mM magnesium acetate, 10 mM potassium phosphate buffer pH 7.2. Ribosomes were labelled as before [l] with 14Clabelled amino acids (CFB 104) and [3H]uridine (Radiochemical Centre, Amersham), except that isotope input was increased t o give specific activities of approximately 450 counts x min-l x pg-l for 14Clabelled protein, and 2500 counts x min-l x pg-l for [SH]RNA. Separation of RNA Protein Fragments Radioactive 30-5 ribosomes were hydrolysed with ribonuclease T, or pancreatic ribonuclease (Sigma) for 4.5 h a t room temperature, in the Vo1.29, No.3,1972
The Dissociation of Escherichia coli Ribosomes
European Journal of Biochemistry, 1973
A systematic study has been made of the dissociation into subunits of the ribosomes of Escherichia coli. By analytical ultracentrifugation, ribosomes were shown to be in dynamic reversible equilibrium with their subunits. This enabled a thermodynamic analysis to be made of the association-dissociation reaction and of several factors which influence it : the concentration of monovalent and divalent cations, the presence of transfer RNA and supernatant factors, the pH, and hydrostatic pressure. The apparent change in free energy for the association in 10 mM sodium phosphate pH 7.0 was-17.4 kcal/mol. Association was accompanied by tthe net binding of mabqesium ions or the net release of monovalent cations (potassium, sodium, or Tris ions). Circular dichroism measurements of ribosomes and their isolated subunits over a range of magnesium and potassium ion concentrations indicated that a change in the conformation of each subunit accompanied association. The change occurring in the 50-S subunit was dependent on the presence of potassium ions, whereas the change in the 30-S subunit was magnesium-ion dependent. The conformational change in the 60-S subunit was critical for the association-dissociation reaction. Under ionic conditions which lead to ribosome dissociation one molecule of transfer RNA was released per 70-S ribosome. This was an independent reaction which did not influence the subunit association-dissociation equilibrium. The release of transfer RNA was accompanied by the net release of four magnesium ions, and occurred with an apparent change in the free energy of 20.8 kcal/mol .
Cell Biochemistry and …, 2005
Three different techniques-light scattering, radiowave dielectric spectroscopy, and fluorescence-were employed to investigate conformational variations in Escherichia coli ribosomes induced by removal of specific proteins. To this end, particles were treated with lithium chloride at different ion strength values to produce ribosomal cores. It was previously observed that treatment of ribosomes to subdenaturing temperatures promotes a structural rearrangement that implies a higher exposure of ribosomal RNA to the solvent. Results presented here strongly suggest that protein elimination from the ribosomal particle produces an overall response recalling the same variation of physical parameters previously observed after thermal treatment. We therefore suggest that high salt treatment produces the same structural modification caused by exposure to subdenaturing temperatures.
Acidic Ribosomal Proteins from Eukaryotic Cells. Effect on Ribosomal Functions
European Journal of Biochemistry, 1979
Precipitation of Saccharomyces cerevisiae ribosomes by ethanol under experimental conditions that do not release the ribosomal proteins can affect the activity of the particles. In the presence of 0.4 M NH4Cl and 50% ethanol only the most acidic proteins from yeast and rat liver ribosomes are released. At 1 M NH4Cl two more non-acidic proteins are lost from the ribosomes. The release of the acidic proteins causes a small inactivation of the polymerizing activity of the particles, additional to that caused by the precipitation itself. The elongation-factor-2-dependent GTP hydrolysis of the ribosomes is, however, more affected by the loss of acidic proteins. These proteins can stimulate the GTPase but not the polymerising activity when added back to the treated particles. Eukaryotic proteins cannot be sustituted for bacterial acidic proteins L7 and L12. We have not detected immunological cross-reaction between acidic proteins from Escherichia coli and those from yeast, Artemia salina and rat liver or between acidic proteins from these eukaryotic ribosomes among themselves.
Characterization of Ribosomal Aggregates Isolated From Liver
Proceedings of the National Academy of Sciences, 1964
Polyribosomal aggregates have been described in mammalian cells, bacteria, and plants.1-6 These aggregates are thought to consist of a linear array of ribosomes attached to a strand of messenger RNA and appear to be the units responsible for protein synthesis.
Analytical Biochemistry, 1979
Thermal shock of intact Escherichia coli, which destroys selectively the 30 S ribosome subunit (A. Weiss and M. Tal, 1973, Biochemistry 12, 4534-4540), has been investigated as a simple procedure to prepare large amounts of pure 50 S ribosome subunits. The structural and functional properties of the 50 S particles prepared from a thermally shocked RNase 1-strain ofE. coli appear to be very similar to those of 50 S ribosomes isolated on sucrose gradients after dissociation of 70 S ribosomes. There was no significant difference in the protein composition of the two ribosome preparations. LiCl-Extracted ribosome cores had similar compositions and their functional activities could be reconstituted with either the homologous or heterologous split protein fractions. The 23 S rRNA of 50 S ribosomes isolated from the RNase 1-strain was essentially intact after the heat treatment. In contrast ribosomes and core particles from RNase + strains had extensively degraded RNA and functional activities could not be reconstituted.