Detection of a membrane-associated protein on detached membrane ribosomes in Staphylococcus aureus

Membrane ribosomes from Staphylococcus aureus which were detached from the membrane by extraction with the nonionic detergent Triton X-100 retained a protein (MBRP) with a molecular weight of 60 000, which was absent from cytoplasmic ribosomes. MBRP was detected and quantified by immunological methods. When membrane ribosomes were dissociated into 50S and 30S subunits, MBRP remained associated with the 50S particle. MBRP was found both on membrane ribosomes and in the cytoplasm in roughly equal amounts. When added to Triton X-100-solubilized protoplasts, antibodies to MBRP produced immunoprecipitates which contained a complex of MBRP and three other proteins with molecular weights of 71 000, 46 000 and 41 000. Four proteins with the same molecular weights as those of the MBRP complex were found associated with membrane ribosomes. The proteins of molecular weight 71 000, 60 000, 46 000 and 41 000 seemed to be present in stoichiometrically equivalent amounts in the complex.     

Studies on structural proteins of the rat liver ribosomes. I. Molecular wights of the proteins of large and small subunits.

Structural proteins of active 60-S and 40-S subunits of rat liver ribosomes were analysed by two-dimensional polyacrylamide gel electrophoresis. 35 and 29 spots were shown on two-dimensional gel electrophoresis of proteins from large and small subunits, respectively. It was noted that the migration distances of stained proteins with Amido black 10B remained unchanged in the following sodium dodecyl sulfate-acrylamide gel electrophoresis, although some minor degradation and/or aggregation products were observed in the case of several ribosomal proteins, especially of those with high molecular weights. This finding made it possible to measure the molecular weight of each ribosomal protein in the spot on two-dimensional gel electrophoresis by following sodium dodecyl sulfate-acrylamide gel electrophoresis. The molecular weights of the protein components of two liver ribosomal subunits were determined by this 'three-dimensional' polyacrylamide gel electrophoresis. The molecular weights of proteins of 40-S subunits ranged from 10 000 to 38 000 and the number average molecular weight was 23 000. The molecular weights of proteins of 60-S subunits ranged from 10 000 to 60 000 and the number average molecular weight was 23 900.     

Quantitative analysis of the protein composition of yeast ribosomes

The molecular weights of the individual yeast ribosomal proteins were determined. The ribosomal proteins from the 40-S subunit have molecular weights ranging from 11 800 to 31 000 (average molecular weight = 21 300). The molecular weights of the 60-S subunit proteins range from 10 000 to 48 400 (average molecular weight = 21 800). Stoichiometric measurements, performed by densitometric scanning on ribosomal proteins extracted from high-salt dissociated subunits revealed that isolated ribosomal subunits contain, besides some protein species occurring in submolar amounts, a number of protein species which are present in multiple copies: S13, S27, L22, L31, L33, L34 and L39. The mass fractions of the ribosomal proteins which were found to be present on isolated ribosomes in non-unimolar amounts, were re-examined by using an isotope dilution technique. Applying this method to proteins extracted from mildely isolated 80-S ribosomes, we found that some protein species such as S32, S34 and L43 still are present in submolar amounts. On the other hand, however, we conclude that some other ribosomal proteins, in particular the strongly acidic proteins L44 and L45 get partially lost during ribosome dissociation. Proteins L44/L45 appears to be present on 80-S ribosomes in three copies.     

The mitochondrial ribosomes of Neurospora crassa. II. Comparison of the proteins from Neurospora crassa mitochondrial ribosomes with ribosomal proteins from Neurospora cytoplasm, from rat liver mitochondria and from bacteria.

1. It has been shown by Datema et al. (Datema, R., Agsteribbe, E. and Kroon, A.M. (1974) Biochim. Biophys. Acta 335, 386--395) that Neurospora mitochondria isolated in a Mg2+-containing medium (or after homogenization of the mycelium in this medium and subsequent washing of the mitochondria in EDTA-containing medium) possess 80-S ribosomes; mitochondria homogenized and isolated in EDTA medium yield 73-S ribosomes. The ribosomal proteins of the subunits of 80-S and 73-S ribosomes were compared by two-dimensional electrophoresis. The protein patterns of the large, as well as of the small subunits are very similar but not completely identical; the most conspicuous difference is that the large subunit of 80 S contains about eight more proteins than the large subunit of 73 S. 2. The contamination by Neurospora cytoplasmic 77-S ribosomes in the 80-S preparations, if present, is only minor. 3. Neurospora cytoplasmic ribosomes contain 31 proteins in the large, and 21 proteins in the small subunit. 4. Neurospora 80- mitochondrial ribosomes contain 39 proteins in the large, and 30 proteins in the small subunit 30 proteins. 5. Rat liver mitochondrial ribosomes contain 40 proteins in the large and at least 30 proteins in the small subunit. About 50% of these proteins has an isoelectric point below pH 8.6. 6. The pattern of Paracoccus denitrificans is very similar to that of other bacterial ribosomes, the large subunit contains 29, the small subunit 18 proteins.     

Protein synthesis defects in temperature-sensitive mutants of Escherichia coli with altered ribosomal proteins

The ribosomes from four temperature-sensitive mutants of Escherichia coli have been examined for defects in cell-free protein synthesis. The mutants examined had alterations in ribosomal proteins S10, S15, or L22 (two strains). Ribosomes from each mutant showed a reduced activity in the translation of phage MS2 RNA at 44 degrees C and were more rapidly inactivated by heating at this temperature compared to control ribosomes. Ribosomal subunits from three of the mutants demonstrated a partial or complete inability to reassociate at 44 degrees C. 70-S ribosomes from two strains showed a reducton in messenger RNA binding. tRNA binding to the 30 S subunit was reduced in the strains with altered 30-S proteins and binding to the 50 S subunit was affected in the mutants with a change in 50 S protein L22. The relation between ribosomal protein structure and function in protein synthesis in these mutants is discussed.     

Studies on the negative circular dichroic bands around 297 nm of ribosomes from bacterial cells.

The small negative CD bands around 297 nm of isolated 30-S and 50-S ribosomal subunits were precisely measured for three bacteria, Bacillus stearothermophilus, Bacillus subtilis and Escherichia coli Q 13. The intensities of the negative CD bands of 30-S subunits were always much greater than those of 50-S subunits irrespective of the bacterial strains, which may be related to the difference in comformations of rRNAs and proteins in the complexes between these subribosomal particles. The dissociation of 70-S ribosomes into two subunits by lowering Mg2+ concentration caused evident enhancement of intensity of the 297 nm CD band, which was completely reversed by the association of the two subunits into 70-S particles. The melting profiles of CD spectra 3 B. stearothermophilus and E. coli were compared and both subunits of the former were found to be more heat stable than those of the latter. It was found that 5 M urea and 0.5% sodium dodecyl sulfate (SDS) treatment caused considerable reduction of the negative CD intensity around 297 nm of 30-S subunits but no significant change of 50-S subunits, while no significant change was observed for the CD spectra of isolated 16-S and 23-S rRNAs by the same treatment. Effects of EDTA treatment and then addition of Mg2+ on the CD spectra and fluorescence emission spectra of the subunits were also observed and the contribution by the interaction between rRNA s and proteins in ribosomes to the small negative band around 297 nm was discussed.     

Specific association of two homologous DNA-binding proteins to the native 30-S ribosomal subunits of Escherichia coli.

The native 30-S ribosomal subunits from Escherichia coli are shown to be associated with two proteins which are different from the known ribosome-associated and ribosomal proteins. Neither protein is foune on native 50-S subunits or on intact ribosomes in the cell extract. The purified proteins re-bind in vitro to free 30-S subunits, but do not bind to either free 50-S subunits or intact ribosomes. The proteins, denoted NS1 and NS2, have been purified and characterized. Both proteins showed the same molecular weight of 9500 by sodium dodecyl sulfate gel electrophoresis but 34 000 by gel filtration. Upon treatment with cross-linking reagents the purified proteins gave higher molecular weight species up to the tetrameric ones showing that they exist in solution as tetramers. The amino acid compositions, tryptic fingerprint patterns and N-terminal sequences of the two proteins have been determined. These data show that NS1 and NS2 possess distinct primary structures but with extensive sequence homology. Antibodies raised against the purified proteins cross-reacted in double immuno-diffusion tests confirming further the homology. Because of the similarity in properties a sample of the DNA-binding protein HD (Berthold, V. and Geider, K. (1976) Eur. J. Biochem. 71, 443--449) was compared to NS1 and NS2. In terms of several criteria, the protein HD is found to be a mixture of two proteins, namely NS1 and NS2. The present report is the first instance of an association of DNA-binding proteins to the ribosome.     

Functional Inactivation and Appearance of Breaks in RNA Chains Caused by Gamma Irradiation of Escherichia coli Ribosomes

70S ribosomes and 30S ribosomal subunits from Escherichia coli MRE 600 were exposed to gamma irradiation at -80szC. Exponential decline of activity with dose was observed when the ability of ribosomes to support the synthesis of polyphenylalanine was assayed. Irradiated ribosomes showed also an increased thermal lability. D₃₇ values of 2.2 MR and 4.8 MR, corresponding to radiation-sensitive molecular weights of 3.1 × 10⁵ and 1.4 × 10⁵, were determined for inactivation of 70S ribosomes and 30S subunits, respectively. Zone sedimentation analysis of RNA isolated from irradiated bacteria or 30S ribosomal subunits showed that at average, one chain scission occurs per four hits into ribosomal RNA. From these results it was concluded that the integrity of only a part of ribosomal proteins (the sum of their molecular weights not exceeding 1.4 × 10⁵) could be essential for the function of the 30S subunit in the polymerization of phenylalanine. This amount is smaller if the breaks in the RNA chain inactivate the ribosome.     

Protein composition of Saccharomyces cerevisiae mitochondrial ribosomes

Protein composition of mitochondrial ribosomes of the yeast Saccharomyces cerevisiae was analysed by two-dimensional electrophoresis. The small (37S) mitoribosomal subunit contains 36 different polypeptides with molecular weights ranging from 10,000 to 60,000. The large (50S) subunit is composed of 41 proteins with molecular weights from 10,000 to 43,000. The molecular weights of mitoribosomal small and large subunits are 1.85 MDa and 2.35 MDa, respectively. Proteins represent 60-62% and 42-45% of the total mass of 37S and 50S subunits respectively. On the basis of the protein content and molecular weights of individual proteins we conclude that all mitoribosomal proteins are present in the mitoribosome in equimolar proportions.     

Isoelectric points and molecular weights of salt-extractable ribosomal proteins.

Rat liver ribosomes prepared in low salt buffer contain basic and acidic proteins not found on ribosomes washed in high salt buffer. Proteins extracted from liver ribosomes by 500 mM KCL were characterized by acid urea-polyacrylamide gel electrophoresis, sodium dodecyl sulfate - polyacrylamide gel electrophoresis and gel isoelectric focusing. The salt-solubilized proteins contain 12 polypeptides with a molecular weight over 67000, several polypeptides with molecular weights less than 67 000, and three polypeptides whose molecular weight exceeded 130 000. Ten to 12 of the proteins were basic, and about 24 acidic proteins were partially or wholly extracted from the ribosomes. Four of the acidic proteins have isoelectric points less than 4.5.     

Ribosomal proteins of pea seeds behaving like anions at pH 8.6]

A group of proteins migrating to the anode at pH 8.6 under polyacrylamide gel electrophoresis was revealed in the total protein of non-dissociated KCl-washed pea seed ribosomes. No proteins with an isoelectric point below pH 4.2 Were found. The presence of acidic proteins in 80 S ribosomes is due to the presence of a specific set of relatively acidic proteins in the total protein of large (5 major and 10 minor components) and small (2 major and 4 minor components) subunits. The mostly acidic proteins are located in the large subunit. The acidic proteins of 60S and 40S subunits are represented by the polypeptide chains with molecular weights from 48 000 to 13 000. The acidic proteins are present in the ribosomes studied in considerably less number than the basic proteins, and the former produce a very weak staining under electrophoretic analysis as compared with the latter. The data obtained suggest that 80S ribosomes of higher plants differ from animal ribosomes by a higher content of relatively acidic proteins.     

Isolation and characterization of free cytoplasmic messenger ribonucleoproteins from rabbit reticulocyte.

Free cytoplasmic messenger ribonucleoproteins have been prepared with a high yield in a zonal rotor. They are rapidly labelled by [3H]uridine. Their sedimentation coefficients are 14.6 S and 19.4 S. Their protein contents are higher than in ribosomes, as indicated by a shift towards greater wavelength in their ultraviolet spectra and a buoyant density of 1.39 g cm-2 in CsCl. They do not cross-react with antiribosome antibodies and their protein patterns in sodium dodecylsulfate and two-dimensional polyacrylamide gel electrophoresis are different from ribosomes'. They may contain up to 30 proteins. The proteins are acidic and their molecular weights range from 22 000 to 100 000. When compared to chromatin they behave more like non-histone proteins than histones.     

Particle weights and protein composition of the ribosomal subunits of the extremely thermoacidophilic archaebacterium Caldariella acidophila.

1. The ribosomal subunits of one thermoacidophilic archaebacterium (Caldariella acidophila) and of two reference eubacterial species (Bacillus acidocaldarius, Escherichia coli) were compared with respect to ribosome mass and protein composition by (i) equilibrium-density sedimentation of the particles in CsCl and (ii) gel-electrophoretic estimations of the molecular weights of the protein and the rRNA. 2. By either procedure, it is estimated that synthetically active archaebacterial 30S subunits (52% protein by wt.) are appreciably richer in protein than the corresponding eubacterial particles (31% protein by wt.) 3. The greater protein content of the archaebacterial 30S subunits is accounted for by both a larger number and a greater average molecular weight of the subunit proteins; specifically, C. acidophila 30S subunits yield 28 proteins whose combined mass is 0.6 X 10(6) Da, compared with 20 proteins totalling 0.35 X 10(6) Da mass for eubacterial 30S subunits. 4. No differences in protein number are detected among the large subunits, but C. acidophila 50S subunits exhibit a greater number-average molecular weight of their protein components than do eubacterial 50S particles. 5. Particle weights estimated by either buoyant-density data, or molecular weights of rRNA plus protein, agree to within less than 2%. By either procedure C. acidophila 30S subunits 1.15 X 10(6) Da mass) are estimated to be about 300 000 Da heavier than their eubacterial counterparts (0.87 X 10(6) Da mass); a smaller difference. 0.15 X 10(6) Da, exists between the archaebacterial and the eubacterial 50S subunits (respectively 1.8 X 10(6) and 1.65 X 10(6) Da). It is concluded that the heavier-than-eubacterial mass of the C. acidophila ribosomes resides principally in their smaller subunits.     

Subunit structure of the mouse epidermal keratin filament.

The two proteins which are the subunits of mouse epidermal keratin filaments have been isolated from fully differentiated epidermis (stratum corneum), viable differentiating cells and cells grown in culture. The proteins have molecular weights of 68 000 and 60 000, consist of families of very similar species, have common N-terminal (N-acetylserine) and C-terminal (glycine) residues, contain 35--40% alpha-helix and are immunologically cross-reacting. In mixtures, the two proteins polymerize in vitro into native-type keratin filaments that are 70--80 angstrom in diameter, up to 30 micrograms long, possess a characteristic alpha-type X-ray diffraction pattern and contain the subunits in the precise molar ratio of 1 : 2 or 2 : 1.     

Studies of basic proteins of 60S- and 40S-subunits of pea seed ribosomes by two-dimensional polyacrylamide gel electrophoresis]

Basic proteins of 60S- and 40S-subunits of pea seed ribosomes were studied by two-dimensional electrophoresis in polyacrylamide gel (PAAG) with subsequent electrophoresis of separated proteins in the gels containing sodium dodecyl sulfate. The proteins under study were found to be electrophoretically heterogenous and showed considerable variations in the staining by amido black and a specific distribution between the two subunits. 47 protein components were detected in the protein preparations of the 60S subunit: 18--as intensively stained, 12--as moderately stained and 17--as weakly stained spots. Presumably, the 60S subunit does not contain proteins whose molecular weights are over 60.000 or below 14.000. Two proteins have mol. weight over 50.000; other proteins have mol. weights varying between 15.000 and 30.000. 32 proteins components were revealed in the protein preparations of the 40S subunit: 15--as intensively coloured, 8--as moderately coloured and 9--as weakly coloured spots. The 40S subunit does not contain proteins whose molecular weights are over 33.000 and below 10.000. Three proteins have mol. weights over 30.000, the other proteins have mol. weights within the interval of 15.000--30.000. The amount of basic proteins in the 80S plant ribosomes is, in all probability, higher as compared to that in animal ribosomes, and this is due to the 60S subunit.     

Comparison of cytoplasmic ribosomal proteins of gateway barley and its mutant

The proteins of the cytoplasmic ribosomes isolated from dry embryos of Gateway barley and its virescens mutant were compared by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). The monosomes of both the lines gave similar patterns with 60 basic proteins. Upon dissociation of the monosomes, for the mutant, the basic proteins of the large subunits migrated more slowly than those of the normal and lacked three proteins but had three additional spots. Also, the proteins of the small subunits differed. The mutant lacked three of the proteins present in the normal but had three additional spots. Therefore, the large and small subunits contained a total of 34 and 41 basic proteins, respectively, in both the lines. There were several spots with identical electrophoretic mobilities in the small and large subunits of these two lines.     

Ribosomal proteins from rabbit reticulocytes: number and molecular weights of proteins from ribosomal subunits.

The ribosomal proteins from 40 S and 60 S subunits of rabbit reticulocytes were separated by two-dimensional polyacrylamide gel electrophoresis. The protein spots stained with Coomassie brilliant blue were cut out and the proteins were extracted. The material extracted from each spot was mixed with proteins of known molecular weight and then analyzed by electrophoresis in polyacrylamide gels containing sodium dodecyl sulfate. Both the total number and the molecular weights of each of the proteins were determined by these procedures. Thirty-two proteins were identified in the 40 S subunits; their molecular weights ranged from 8000 to 39,000 (average mol. wt = 25,000). Thirty-nine proteins were identified in the 60 S subunit; their molecular weights ranged from 9000 to 58,000 (average mol. wt = 31,000). The sum of the molecular weights of the individual proteins from each subunit is in agreement with previous estimations, derived from physico-chemical measurements of the total protein in mammalian ribosomal subunits. The molecular weight distribution obtained for the isolated proteins was nearly identical to that derived from spectrophotometric analysis of polyacrylamide-sodium dodecyl sulfate gels of the total protein mixtures from each subunit stained with Coomassie brilliant blue. The results are consistent with the hypothesis that reticulocyte ribosomes contain one copy of most of their protein constituents.     

A study of mitochondrial ribosomes from the higher plant Solanum tuberosum L.

Ribosomal subunits are isolated from potato tuber mitochondria devoid of contaminating organelles. The sedimentation constants of the two mitochondrial ribosomal subunits are 33S and 50S respectively. The apparent sizes of the high molecular weight RNAs are 19S and 25S.The proteins of these ribosomes have been analyzed by two-dimensional electrophoresis in SDS polyacrylamide gels to determine their number and molecular weights. The small subunit contains 35 protein species ranging from 8 to 60 kDa. The 50S large subunit contains 33 protein species ranging from 12 to 46 kDa. These preliminary results are the first analysis made on mitochondrial ribosomes from a higher plant.     

Nature of conformational restrictions in nuclear superhelical DNA from thymus lymphocytes

The lymphocyte nucleoids of mouse thymus contain about 40% of rapidly labelled nuclear RNA, about 9% of total intracellular protein and all nuclear DNA. Relaxation of superhelical DNA after thymocyte nucleoids treatment with pronase or RNAase suggests that non-histone proteins and/or RNAs are involved in conformational restrictions in the superhelical domains of cell DNAs. Thymocyte nucleoids proteins are represented by two groups of nonhistone proteins with molecular weights of 50 000-60 000 and 75 000-85 000. An essential role in the appearance of conformational restrictions in thymocyte superhelical DNA belongs to disulfide bonds.     

The Chloroplast and Cytoplasmic Ribosomes of Euglena: II. Characterization of Ribosomal Proteins

Cytoplasmic and chloroplast ribosomal proteins were isolated from Euglena gracilis and analyzed on polyacrylamide gels. Cytoplasmic ribosomes appear to contain 75 to 100 proteins ranging in molecular weight from 10,200 to 104,000, while chloroplast ribosomes appear to contain 35 to 42 proteins with molecular weights ranging from 9,700 to 57,900. This indicates that the cytoplasmic ribosomes are similar in composition to other eucaryotic ribosomes, while chloroplast ribosomes have a protein composition similar to the 70S procaryotic ribosome. The kinetics of light-induced labeling of cytoplasmic ribosomal proteins during chloroplast development has been determined, and the results are compared with the kinetics of ribosomal RNA synthesis.