Kazuo Terao

Differences between the protein moieties of active subunits and EDTA-treated subunits of rat liver ribosomes with specific references to a 5 S rRNA - protein complex.

When active 40 S subunits of rat liver ribosomes were treated with EDTA, the conversion of 40 S subunits to 30 S subunits occurred with partial release of 13 kinds of proteins out of 29 kinds of 40 S proteins. Buy contrast, 60 S subunits completely lost one protein (L3) by EDTA-treatment with concomitant release of the fraction sedimenting at about 7 S (7 S fraction). It was found that the 7 S fraction contained 5 S ribosomal RNA as well as L3 protein having a molecular weight of 38,000. Buoyant density in CsCl of the 7 S fraction was 1.60 g/cm3, suggesting that this fraction consisted of RNA and protein at an approximately equal ratio on a weight basis. These findings, taken together with the molecular weights of 5 S rRNA (40,000) and L3 protein, may indicate that the 5 S ribosomal RNA - protein complex from rat liver 60 S subunits consists of one molecule of 5S ribosomal RNA and one molecule of L3 protein.

Identification of neighbouring protein pairs in the rat liver 40-s ribosomal subunits cross-linked with dimethyl suberimidate

(1) The 40-S ribosomal subunits of rat liver were treated with a bifunctional cross-linking reagent, dimethyl suberimidate. Cross-linked protein-protein dimers were separated by two-dimensional acrylamide gel electrophoresis. The stained cross-linked complexes within the gel were radioiodinated without the elution of proteins from the gel and were cloven into the original monomeric protein constituents by ammonolysis. The proteins in each dimer were finally identified by two-dimensional acrylamide gel electrophoresis of the cloven monomeric proteins, followed by radioautography of the stained gel. (2) The molecular weights of cross-linked complexes were determined by SDS-polyacrylamide gel electrophoresis and were compared with those of their constituent proteins. (3) The following dimers were proposed from these results: S3-S12 (S3 or S3a-S11), S4-S12 (S3b-S11, S5-S7 (S4-S6), S5-S22 (S4-S23 or S24), S6-S8 (S5-S7), S8-S16 (S7-S18), S17-S21 (S16--S19) and S22A-S22B (S23-S24), designated according to our numbering system [1]. The designations according to the proposed uniform nomenclature [2] are described in parentheses.

Studies on the small subunit of rat liver ribosomes: Some biochemical properties with specific references to the reconstruction of the small subunit

Abstract 1. The chemical analysis and CsCl buoyant density gradient centrifugation showed that the protein content of the 32-S ribosomal subunit, prepared from rat liver ribosomes by EDTA treatment, was lower than that of the active 40-S subunit, prepared by incubation with a high concentration of KCl. The same analyses of the EDTA-treated 47-S subunit and the active 60-S subunit showed similar protein contents between two types of ribosomal large subunits. The EDTA-treated 32-S subunit prepared in the presence of the liver ribonuclease inhibitor showed poly(U)-binding activity comparable to the active 40-S subunit, although the activity of poly(U)-dependent polyphenylalanine synthesis was very low. 2. Protein-depleted particles (40-S core particles) and split proteins were prepared from the active 40-S subunit by treatment with 0.4 M LiCl in the presence of 5 mM MgCl 2 . About 25 % of protein was dissociated from the active 40-S subunit by this treatment. The selective dissociation of proteins from 40-S subunit was shown by acrylamide gel electrophoresis of split proteins and 40-S core particles. The reconstructed particles from core particles and split proteins from the active 40-S subunit showed definite activity in poly(U)-dependent polyphenylalanine synthesis in conjunction with the active 60-S ribosomal subunit. 3. By incubation of 18-S ribosomal RNA with proteins of the 40-S ribosomal subunit, particles with s value of about 32 S were reconstructed. The protein content of the 32-S reconstructed particles was somewhat lower than that of the EDTA-treated 32-S subunit and acrylamide gel electrophoresis of the proteins of the particles showed a complete set of protein components of the original 40-S subunit, although the densitogram showed a difference in some bands between the 40-S subunit and the reconstructed particles. The reconstructed 32-S particles showed definite activity in poly(U)-binding, but their activity in poly(U)-dependent poly-phenylalanine synthesis in conjunction with the active 60-S sbunit was very low.

Characterization of the proteins of the small subunits of rat liver ribosomes

Abstract Proteins extracted from the small subunits of EDTA-treated rat liver ribosomes with 67% acetic acid were resolved into 19 fractions by CM-cellulose column chromatography, which were further fractionated by gel filtration on Sephadex G-200. From the results of split acrylamide gel electrophoresis of these protein fractions, it is proposed that there are 28 proteins in the small subunits of EDTA-treated liver ribosomes, 12 of these proteins have been isolated in relatively pure states by the gel filtration subsequent to CM-cellulose column chromatography. The molecular weights of most of the proteins range from 15 000 to 40 000 and the sum of the molecular weights of the 28 proteins is about 690 000. That the small subunits of rat liver ribosomes had a higher protein content than that of Escherichia coli may be explained both by the greater number of protein species contained and by the larger average molecular weights of these proteins.

Analytical methods for ribosomal proteins of rat liver 40 S and 60 S subunits by "three-dimensional" acrylamide gel electrophoresis.

Publisher Summary The chapter discusses the analytical methods for ribosomal proteins of rat liver 40 S and 60 S subunits by three dimensional acrylamide gel electrophoresis. During analysis of the proteins of 60 S and 40 S subunits of rat liver ribosomes by two-dimensional acrylamide gel electrophoresis, it was found that the mobility of several proteins by SDS-acrylamide gel electrophoresis remained unchanged even after staining these proteins with Amido Black 10B. Therefore, it was thought to be possible to identify the individual ribosomal proteins and estimate their molecular weights by using two-dimensional acrylamide gel electrophoresis followed by SDS acrylamide gel electrophoresis (three-dimensional electrophoresis). The reason that ribosomes are known to absorb soluble proteins, it is necessary to use purified ribosomes or their subunits free from contamination with cell sap proteins. The chapter also describes methods of preparing pure 40 S and 60 S ribosomal subunits almost free from contamination from cell sap proteins.

Studies on the biosynthesis of structural proteins of liver ribosomes: I. The incorporation of labelled amino acids into basic structural proteins of liver ribosomes in vivo

Abstract In an attempt to elucidate the metabolic properties of the basic structural proteins of liver ribosomes, rat-liver ribosomes were labelled with 14 C-labelled amino acids in vivo . The ribosomes were freed from the nascent protein fraction by EDTA treatment and basic structural proteins were extracted by acetic acid. The labelled basic proteins were subjected to CM-cellulose chromatography to give Fraction I protein which was further fractionated by starch-gel electrophoresis. 1. 1. Starch-gel electrophoresis of Fraction I labelled for 10 min, 2 h and 12 h, showed that this fraction consisted of proteins with different metabolic rates and that slow-moving (less basic) proteins, were in general more active than fast-moving (more basic) proteins. 2. 2. Two-dimensional starch-gel electrophoresis of Fraction I resulted in the separation of this fraction into about 30 protein components. The finding that the incorporation of the label occurred in the majority of the protein spots and was negligible in other areas, provides strong evidence that the bulk of the basic structural proteins undergo metabolic renewal. The heterogeneity in metabolic rate among these proteins was further demonstrated. 3. 3. The time course of the specific activity of Fraction I, labelled in vivo for various time intervals, showed that the rate of incorporation into Fraction I is rather slow, reaching the maximum about 6 h after the injection. In contrast, the incorporation into nascent proteins was very rapid, reaching a maximum at 10 min. 4. 4. The semi-logarithmic plots of the decreasing specific activities exhibited by Fraction I and other cellular components showed that the average renewal rate of the basic structural protein of ribosomes is comparable to that for the total proteins of mitochondria, nucleus and cell sap.

Cross-linking of L5 protein to 5 S RNA in rat liver 60-S subunits by ultraviolet irradiation.

After rat liver 60-S ribosomal subunits were irradiated with ultraviolet light at 254 nm, they were treated with EDTA and then subjected to sucrose density-gradient centrifugation to isolate 5 S RNA-protein complex. When 5 S RNA-protein was analyzed by SDS-acrylamide gel electrophoresis which dissociated noncovalent 5 S RNA-protein, two protein bands were observed. The one showed a slower mobility than the protein band (L5) of 5 S RNA-protein from non-irradiated 60 S subunit and the other showed the same mobility as L5 protein. Since the former band was shown to be specific to ultraviolet-irradiation, it was considered as cross-linked 5 S RNA-protein. After the two protein bands were iodinated with 125 I, labeled protein was extracted and treated with RNAase. Thereafter, it was analyzed by two-dimensional acrylamide gel electrophoresis, followed by autoradiography. The results indicate that the protein component of cross-linked 5 S RNA-protein is L5 protein (ribosomal protein; these proteins are designated according to the proposed uniform nomenclature. The correlation between that and our nomenclature was reported by McConkey et al. (1979) Mol. Gen. Genet. 169, 1-6. They also confirm the results previously reported (Terao, K., Takahashi, Y. and O(gata, K. (1975) Biochim. Biophys. Acta 402, 230-237).

Studies on the biosynthesis of structural proteins of liver ribosomes. II. The incorporation of labelled amino acid into basic structural proteins of liver ribosomes by a cell free system.

Abstract 1. A cell-free system, consisting of rat-liver polysomes and cell sap, incorporated [14C]amino acids into a basic protein fraction (Fraction I) of liver ribosomes. The Fraction I was prepared by CM-cellulose chromatography of the acetic acidsoluble fraction of ribosomes freed from nascent proteins. 2. The distribution pattern of the radioactivity in starch-gel electrophoresis of the labelled Fraction I was examined. A higher activity was generally associated with the slower-moving proteins. The activity was rather low in the fast-moving proteins. 3. The labelled Fraction I was fractionated into two components by Sephadex G-200 column chromatography. Component I, eluted first, had higher specific activity than Component II which was eluted later. On the subsequent starch-gel electrophoresis, Component I gave a slow-moving and rather diffuse pattern and a high radioactivity was found near the origin. Component II gave distinct fast-moving protein bands and some of the radioactivity was associated with these protein bands. 4. Two-dimensional starch-gel electrophoresis of the labelled Component II revealed that the radioactivity was incorporated into most of the protein spots and no significant activity was found in other areas. The distribution of the radioactivity in each spot was not uniform. 5. It is proposed that a considerable part of “basic structural proteins” of liver ribosomes are synthesized on polysomes through the general protein-synthetic pathway involving amino acyl-soluble RNA as the intermediate.

[42] Analytical methods for synthesis of ribosomal proteins by cell-free systems from rat liver

Publisher Summary This chapter describes the analytical methods for synthesis of ribosomal proteins by two kinds of cell-free systems from regenerating rat liver: postmitochondrial supernatant as well as various kinds of polysomes and cell sap. The former has initiating activity although it is rather low. Ribosomal proteins are purified by acetic acid extraction followed by CM-cellulose column chromatography. These procedures separate ribosomal proteins from the bulk of cell sap proteins. To examine the incorporation into individual ribosomal proteins, fraction I from CM-cellulose chromatography is subjected to two-dimensional acrylamide gel electrophoresis. The radioactivity of stained proteins on two-dimensional gel is then measured. The yield of ribosomal proteins during the purification procedures is variable, it is recommended to use double-labeling techniques to obtain quantitative data on the incorporation into ribosomal proteins or to compare the activities of different kinds of polysomes for biosynthesis of ribosomal proteins. The disadvantage of the chromatographic procedures described in the chapter is that several kinds of ribosomal proteins, including the less basic proteins are lost. To examine the incorporation into these proteins, three-dimensional electrophoresis of acetic acid-soluble protein is available and is described in the chapter.

An ATPase Center of Rat Liver 30S-5SRNP Particles

Treatment of 30S-5SRNP with 1 M Cs(2)SO(4) at 2 degrees C overnight followed by sucrose density-gradient centrifugation yielded particles smaller than 30S-5SRNP, designated as CsS-particles. CsCl density-gradient centrifugation of CsS-particles showed the homogeneity of the particles containing about half the amount of proteins in 30S-5SRNP particles. The particles contained 18SrRNA, 5SRNP and about half the number of proteins in 30S-5SRNP. The ATPase activity of freshly prepared CsS-particles was about half the original 30S-5SRNP level although it was unstable even at 2 degrees C. Poly(U) slightly enhanced the activity, and phe-tRNA(phe) stimulated it concentration-dependently. EF-1a alone enhanced it, and in combination with poly(U) and phe-tRNA(phe) stimulated it markedly. EF-2 alone markedly increased it. The activity with the full components for elongation described above became very high, being comparable to that of the original 30S-5SRNP and twice that of 40S subunits. A two-dimensional electrophoretogram of the protein in CsS-particles revealed 9 small subunit protein species, in addition to L5, which included proteins interacting with mRNA and two elongation factors. Taken together with the results of our preceding study indicating the participation of ATPase of 80S ribosomes in peptide elongation, the present results indicate CsS-particles may be a part of the ATPase centre of 80S ribosomes.