Ribosomes of rat liver catalyze ‘minimal’ donor reaction (original) (raw)
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Structure and Function of Rat-Liver Ribosomes. Modification by 2Methoxy5-nitrotropone Treatment
European Journal of Biochemistry - EUR J BIOCHEM, 1976
Rat liver ribosomes and 60-S ribosome subunits were treated with the primary-amino-group-specific reagent 2-methoxy-5-nitrotropone. Important differences in the sensitivity of several ribo- somal activities to inactivation by the reagent were observed. While elongation-factor-dependent activities are totally abolished in the treated particles, peptidyl transferase activity is either unaffected in 60-S ribosomal subunits or even strongly stimulated in 80-S ribosomes. Analysis of the ribosomal proteins modified by nitrotropone made it possible to draw some conclusions on their accesibility in the ribosomal structure and to relate some proteins with their involvement in the ribosome active centers. Thus, proteins L3, L13, L15 and L23 seem to be in a rather well protected position while proteins L10, L35, L37, X1 and X2 are totally exposed to the reagent. The protein accessibility also depends on the ribosome conformation, proteins L14 and L17, for instance, being sensitive in 80-S ribosomes and protected in 60-S subunits. In relation to the implication of proteins in functional centers, the data presented here together with other data obtained from protein-deficient core particles seem to indicate a possible role of proteins L21 and/or L26 in the peptidyl transferase center.
Free and membrane-bound ribosomes in rat liver
The Journal of biological chemistry, 1967
Ribosomes in rat liver are present either free or attached to endoplasmic reticulum and in both states exist largely as polyribosomal aggregates. The present communication presents a quantitative study of the distribution of ribosomes between these two states and examines a number of properties of these two groups of ribosomes. It is shown that the base composition and the rate of synthesis of the ribosomal ribonucleic acid of free and membrane-bound ribosomes are the same, and these tindings, along with the results of previous studies of other properties, are consistent with the possibility that there are no intrinsic differences between the ribosomes found in these two states. Since certain conditions are known to produce alterations in the distribution of free and membrane-bound ribosomes which are rapid with respect to the turnover time of ribosomal RNA, it is further suggested that a given ribosome can exist in either state according to the needs of the cell. Although the administration of hydrocortisone in uivo induces acute rises in the levels of certain specific liver enzymes within a few hours, it has no marked effect upon the ratio of free to membrane-bound ribosomes during this period. The ribosomal aggregates obtained by the methods used here are shown to be sufficiently pure to permit the use of ultraviolet absorption as a direct measure of their concentration without applying a correction for ferritin absorption. Homogenization and zone centrifugation do not produce significant artifacts in the determination of the proportions of free and membrane-bound ribosomes. In a previous communication it was shown that the great majority of ribosomes in rat liver are attached to large membranous structures (1). Data were presented which showed that ribosomes in all fractions of liver homogenates are active in protein synthesis in tivo and in vitro, and it was further shown
Peptidyl Transferase Center of Rat-Liver Ribosome Cores
European Journal of Biochemistry, 1977
Protein-deficient particles have been obtained by treating rat liver 80-S ribosomes or their 60-S subunits with 1 M NH4Cl in the presence of 50% ethanol at 0°C (P0-cores) and 37°C (P37-cores). The P0-cores from 80-S ribosomes are totally inactive in polyphenylalanine synthesis but fully active in the ‘fragment assay’ to test peptidyl transferase activity. The polymerizing activity of the cores is restored up to 40–50% of control activity by incubation in the presence of the split proteins. Three proteins are totally lost in the treatment, namely proteins L12, L40/41 and S25. A series of up to nine different spots in the region of the L40/41 proteins are detected when the split fraction is analyzed by two-dimensional electrophoresis. This series of spots is, however, reduced to only two proteins when the second dimension is carried out in the presence of sodium dodecylsulphate.80-S ribosome-derived P37-cores are about 80% active in the ‘fragment reaction’ while 60-S-subunit-derived particles are inactive in this assay. The inhibitory effect of a number of antibiotics is differentially affected by the treatment suggesting different localization of their binding sites. A comparative study of the proteins released by treatment in the two types of particles suggests the involvement in the peptidyl transferase center of the ribosome of one or more of the following proteins: L21, L24, L27, L28 and L36.
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.
European Journal of Biochemistry, 1978
Eucaryotic L7/L12-type proteins are present in ethanol/salt extracts (P1 protein) of ribosomes from Artemia salina and rat liver. These proteins are partially phosphorylated and occur in two forms of closely related structure: a major form eL12 havkg methionine at the N-terminal position and a minor form of eL12 (eL12') which seems slightly elongated and contains a blocked N terminus. Purification of the four different forms of this protein, eL12, eL12-P, eL12' and eL12'-P, was performed by ion-exchange chromatography on carboxymethyl-cellulose and DEAEcellulose. Using a radioimmuno assay, 1.8 copies of eL12 and 0.9 of eL12' were found on the 80-S A. salina ribosome. In ribosomes of both rat liver and A . salina, eL12 is present in a larger quantity than eL12'. 40-S and 60-S ribosomal subunits extracted with ethanol/salt were essentially free of eL12 proteins. A large pool of eL12 was found in the cytosol after removal of the ribosomes by centrifugation or molecular sieving. The proteins of rat liver and A . salina are similar with regard to their isoelectric points and molecular weights. Sedimentation equilibrium studies indicated that the isolated protein eL12 occurs as a dimer. 6 0 3 ribosomal subunits from several eucaryotic organisms contain an acidic protein with properties which resemble the protein L7/L12 from bacterial 50-S ribosomal subunits . In eucaryotes there are also two related forms of the protein. The difference between them is not due to acetylation as in Escherichia coli but rather phosphorylation .
European journal of biochemistry / FEBS, 1976
Protein-deficient ribosomal particles obtained by treatment of 50-S subunits from Escherichia coli ribosomes with 1 M NH4Cl and 50% ethanol contain less than 3% of proteins L7 and L12 and about 7% of proteins L10 and L11. Proteins L1, L5, L8/9 and L25 are also released during the treatment but in amounts accounting for less than 40%. The particles are able to form peptide bonds in different systems, such as 'fragment reaction', puromycin reaction and formation of dipeptides. They also bind N-acetylphenylalanyl-tRNA and phenylalanyl-tRNA non-enzymically but are unable to support any of the elongation-factor-dependent reactions tested. However, when methanol is present, they display up to 20% of the control EF-G-dependent GTP activities such as GTP hydrolysis and formation of the ternary complex EF-G-GuoPP(CH2)P-ribosome. The first activity is totally sensitive to the antibiotic thiostrepton while the formation of the ternary complex is unaffected by the drug. When measured by...
Electron Microscopic Observations on the Large Subunit of the Rat Liver Ribosome
The Journal of Cell Biology, 1970
Active large subunits obtained by urea treatment of rat liver ribosomes, 59S, were compared with large subunits in intact ribosomes and with the 50S subunits obtained by EDTA treatment. For electron microscopy the specimens were negatively stained or shadow cast. The negatively stained 59S subunits had a slightly ovoidal form; their average dimensions, 244 ± 17 x 207 ± 18 A, were very close to the dimensions of the large subunits in intact ribosomes, and lay between the theoretical dimensions for anhydrous and fully hydrated particles that were calculated from the physical properties of the subunits in solution. The shadow-cast preparations showed particles of similar shape. The 50S subunits, which had lost their 5S RNA, were shadow cast at the same time. They appeared to be more spread out than the 59S subunits and had threadlike extensions. In the positively stained regions of uranyl oxalate-stained preparations the 50S particles varied greatly in shape and size, with average dime...