Showing papers on "Ribosomal protein published in 1967"

The formation, distribution and function of ribosomes and microsomal membranes during induced amphibian metamorphosis.

Abstract: 1. A lag period of about 4 days preceded the onset of metamorphosis precociously induced by tri-iodothyronine in tadpoles of the giant American bullfrog (Rana catesbeiana). It was established by the accelerated synthesis or induction of carbamoyl phosphate synthetase and cytochrome oxidase in the liver, serum albumin and adult haemoglobin in the blood, acid phosphatase in the tail, and the increase in the hindleg/tail length ratio. 2. A 4- to 6-fold stimulation, 2 days after the induction of metamorphosis, of the rate of synthesis of rapidly labelled nuclear RNA in liver cells was followed by an increasing amount of RNA appearing in the cytoplasm. Most of the newly formed RNA on induction of metamorphosis was of the ribosomal type. An accelerated turnover at early stages of development preceded a net accumulation of RNA in the cytoplasm, with no change in the amount of DNA per liver. 3. Most hepatic ribosomes of the pre-metamorphic tadpoles were present as 78s monomers and 100s dimers; metamorphosis caused a shift towards larger polysomal aggregates with newly formed ribosomes that were relatively more tightly bound to membranes of the endoplasmic reticulum. 4. The appearance of new polyribosomes in the cytoplasm on induction of metamorphosis was co-ordinated in time with a stimulation of synthesis of phospholipids of the smooth and rough endoplasmic reticulum, followed by a gradual shift in preponderance from the smooth to the rough type of microsomal membranes. 5. Electron- and optical-microscopic examination of intact hepatocytes revealed a striking change in the distribution and nature of ribosomes and microsomal membranes during metamorphosis. 6. Ribosomes prepared from non-metamorphosing and metamorphosing animals were identical in their sedimentation coefficients and in the structural ribosomal proteins. The base composition and sedimentation coefficients of ribosomal RNA were also identical. Induction of metamorphosis also did not alter the incorporation of 32P into the different phospholipid constituents of microsomal membranes. 7. Nascent 14C-labelled protein with the highest specific activity was recovered in the `heavy' rough membrane fraction of microsomes, whereas little 14C was associated with `free' polysomes. Protein synthesis in vivo was most markedly stimulated during metamorphosis in the tightly membrane-bound ribosomal fraction after the appearance of new ribosomes. 8. The rate of synthesis of macromolecules in vivo could not be followed beyond 7–8 days after induction because of variable shifts in precursor pools due to regression of larval tissues. 9. The stimulation of RNA and ribosome formation was specifically associated with the process of metamorphosis since no similar response to thyroid hormones occurred in those species (Axolotl and Necturus) in which the hormones failed to induce metamorphosis.

Mammalian Ribosomal Protein: Analysis by Electrophoresis on Polyacrylamide Gel

Abstract: Ribosomal protein from five mammalian tissues when analyzed by discontinuous electrophoresis on polyacrylamide gel at pH 4.5 yielded 24 bands. Densitometric tracings indicated that the patterns of the basic ribosomal proteins from the several tissues were qualitatively similar. Protein from Escherichia coli ribosomes analyzed at pH 4.5 gave 29 bands, and the pattern was different from that of mammalian ribosomal protein. No distinct band was found when mammalian ribosomal protein was analyzed at pH 8.3 (acidic proteins). Ribosomal protein from Escherichia coli gave eight bands at pH 8.3. Thus, the structure of the genes responsible for synthesis of ribosomal protein in several mammalian tissues is the same, and different genes direct synthesis of ribosomal protein in bacteria.

Ribosomal proteins of Escherichia coli. I. Demonstration of different primary structures

Abstract: Ribosomal protein from E. coli separates during gel electrophoresis into a large number of components.'-3 A variety of experiments have been done to determine if the apparent complexity might be misleading and be caused by interactions among a smaller number of components. No evidence has been found in favor of this hypothesis.\"' 3-7 The most likely conclusion is that the multiple components seen on gel electrophoresis correspond to different protein molecules in the ribosome population. The possibility remains, however, that the different electrophoretic components are closely related chemically and are only slight modifications or combinations of one or a few primary structures. This view has been favored by success in elucidating the general mechanism of protein synthesis while considering the ribosome a passive structure or template, and by the plausible analogy between ribosome and virus structure. On the other hand, the recent demonstration that specific fractions of ribosomal protein restore in vitro activity to protein depleted particles6-9 and the evidence indicating that peptidyl transferase is an integral part of the 50S ribosome'0'-2 are more consistent with the concept of the ribosome as an enzyme complex composed of functionally specific, chemically distinct proteins. The experiments presented here were designed to distinguish between these alternatives using two approaches. (1) The number of tryptic peptides of the total protein containing sulfur was examined. The relationship of this number to the total calculated number of sulfur-containing residues should reflect the extent to which common sequences occur. (2) Several pure proteins have been isolated and tryptic fingerprints and amino acid compositions compared. Evidence of both types leads to the conclusion that the ribosomal proteins are chemically heterogeneous. Materials and Methods.-Early and late log phase E. coli B cells were purchased from General Biochemicals, Chagrin Falls, Ohio. Cell extracts were prepared by homogenization with glass beads\"3 or with a Hughes pressure cell. Ribosomes and ribosomal subunits were prepared by standard methods.\"' 15 Ribosomal proteins were extracted with 66 per cent acetic acid.'6 Protein concentrations were determined by the Folin method.'7 N-terminal groups were found by the dinitrofluorobenzene (DNFB) method.'8 19 Acrylamide gel electrophoresis and radioautography were done as described by Traut.3 S36 MRE-60021 cells were grown as described previously.3 Cysteine was labeled selectively by including 10 ,g/ml of nonradioactive methionine in the medium. The exclusion of S35 from methionine was confirmed by analysis of acid hydrolysates by paper electrophoresis at pH 1.9 followed by radioautography. Carboxymethyl cellulose was obtained from Calbiochem (Los Angeles) and from W. and R. Balston, Ltd. (England). Performic acid oxidation was carried out according to Nathans1° and carboxymethylation according to Spackman et al.26 Purification of the proteins was achieved on carboxy-

Ribosomal proteins. I. Isolation, amino acid composition, molecular weights and peptide mapping of proteins from E. coli ribosomes.

Abstract: SummaryThe proteins of E. coli ribosomes were separated by a specially developed type of preparative polyacrylamide gel electrophoresis and proteins corresponding to 16 of the separated bands have so far been isolated. The amino acid compositions and the tryptic peptide maps of these proteins show certain degree of similarity as well as distinct differences. Determination of molecular weights revealed a wide range: The lowest molecular weight was 9,000 and the highest 41,000 for the proteins so far studied.The similarities between various ribosomal proteins in their amino acid compositions and their peptide maps on one hand and the wide range in their molecular weights on the other hand can be explained by a hypothesis involving gene duplications.

Structure and function of E. coli ribosomes, IV. Isolation and characterization of functionally active ribosomal proteins.

Abstract: E. coli ribosomes structure, molecular components, function and protein synthesis with artificial ribosome derivatives deficient in one component

Optical rotatory dispersion of E. coli ribosomes and their constituents.

Abstract: Investigations have been made on the optical rotatory dispersion properties of E. coli ribosomes and their constituent RNA and proteins. The results indicate that (1) no conformational changes are involved in the formation of a 70S particle from the 50S and 308 subunits, (2) the E. coli ribosomal proteins are similar to most globular proteins with little α-helix content, and (3) the conformation of RNA and proteins inside the ribosome is very similar to that in the free state.

Ribosomal proteins. II. A rapid micromethod for electrophoretic separation of Escherichia coli ribosomal protein on gelatinized cellulose acetate.

Abstract: SummaryA new method for electrophoretic separation of ribosomal proteins on gelatinized cellulose acetate is presented. The main features of this method are: 1. fast performance, 2. five to ten micrograms protein are sufficient, 3. the separation of the proteins is mainly due to their net charge, 4. quantitation of separated proteins by densitometry or autoradiography of the original strips is possible. In addition to the described difference in the electrophoretic patterns of E. coli K12 and E. coli B ribosomal proteins in disc polyacrylamide gel electrophoresis (Leboy et al., 1964) some further differences are detected by the CAG electrophoresis.

Ribosomes and Ribosomal Protein from Neurospora crassa I. Physical, Chemical, and Immunochemical Properties

Abstract: Ribosomes from Neurospora crassa, initially characterized by ultracentrifugal and immunochemical analyses, have been used to prepare ribosomal protein for physical, chemical, and immunochemical study. The acrylamide gel disc electrophoretic profiles of Neurospora ribosomal protein exhibit a degree of heterogeneity comparable to what has been observed in other systems. Only by chemical modification or by aggregation of the protein do alterations in the profile become apparent. Disulfide-bond formation appears to play a role in the aggregation of ribosomal protein to complexes of S20,w = 200. The aggregation can be prevented by alkylation of −SH groups, and protein treated in this fashion has a subunit molecular weight of about 20,000 as determined by equilibrium centrifugation. Finger-printing of tryptic peptides indicates that more than one unique sequence of amino acids must be present in ribosomal protein, although gross primary structural heterogeneity is questioned. Antigenic heterogeneity is much less apparent; only a few precipitin bands are resolved by immunodiffusion tests, although complete reactivity of total ribosomal protein is suggested by quantitative precipitin analysis. The antigenically active ribosomal protein components appear to reside in at least two fractions; one is removed readily from the ribosome by CsC1 treatment. Ribosomal protein of N. crassa possesses antigenic determinants present in E. coli ribosomal protein as judged by spur formation in immunodiffusion tests. Images

Magnesium Starvation of Aerobacter aerogenes III. Protein Metabolism

Abstract: The metabolism of the ribosomal and soluble protein components of Aerobacter aerogenes was examined during its incubation in a Mg++-deficient medium. Bacteria were exposed to leucine-H3 during the exponential growth period preceding Mg++ starvation, and extracts were prepared after intervals of starvation and were centrifuged through gradients of sucrose to separate ribosomal from soluble proteins. Ribosomal proteins synthesized during the preceding exponential growth were slowly lost from the ribosomes; after 8 hr of starvation, few, if any, sedimented with ribosomes. Losses of total protein, together with the known rate of ribosome decay during Mg++ starvation, suggested that these ribosomal proteins are ultimately degraded to acid-soluble products and account for all protein lost by the starving cells. These conclusions were supported by studies of Mg++ starvation in a uracil-requiring strain of A. aerogenes: during uracil starvation a smaller fraction of the proteins synthesized were ribosomal, and the fraction of protein which subsequently decayed during Mg++ starvation was correspondingly less. During recovery from Mg++ starvation, proteins, lost from disintegrated ribosomes, were not detectably reutilized into new particles even before their degradation to acid-soluble products was complete. Synthesis of soluble proteins continued for more than 24 hr of starvation at a rate per milliliter close to 45% of the instantaneous rate per milliliter of the exponentially growing bacteria at the time Mg++ was removed. This value agreed with that found previously for synthetic rates of deoxyribonucleic acid, transfer ribonucleic acid, and ribosomal ribonucleic acid during starvation relative to rates during exponential growth.

Primary structure heterogeneity in ribosomal proteins from Escherichia coli.

Abstract: FUNCTIONAL heterogeneity of ribosomal proteins from Escherichia coli is extensive and in vitro polypeptide synthesis requires at least six different protein fractions1. The structural heterogeneity of ribosomal proteins is even more impressive; on gel electrophoresis of 70S ribosomal protein from E. coli about thirty protein bands separate and opinion favours the presence of at least as many distinct proteins2–10. Little is known, however, about the extent to which the primary structure of these proteins is related. In this communication a comparison between the amino-acid composition and tryptic fingerprints of eight simplified protein fractions, isolated from 70S ribosomal proteins of E. coli, will be made. In addition the purification and some properties of three single ribosomal proteins, one acidic and two basics, will be presented. Fig. 1 gives the results of applying 1 g 70S ribosomal protein (prepared using glacial acetic acid2,10) to a column of CM cellulose. Four different peaks were reproducibly resolved. The electrophoresis patterns on polyacrylamide gels (15 per cent) of selected fractions are shown in Fig. 2. As Table 1 shows, the amino-acid compositions of these fractions, determined on a Technicon autoanalyser, are quite distinct. Each fraction contains at least one amino-acid, the percentage of which is substantially greater or less than the percentage of that amino-acid in any other fraction. The relative difference in the contents of basic over acidic amino-acid residues increases regularly from a value of −7 mole per cent to 12 mole per cent. Tryptic fingerprints confirm the presence of extensive differences in primary structure between each of these fractions; with the exception of fractions 1a and 1b, each fraction shows a different tryptic fingerprint with strong spots uniquely missing or present on comparison with any of the other fingerprints (Fig. 3).

Properties of ribosomal proteins from two mammalian sources.

Abstract: 1. Ribosomal protein fractions from rabbit reticulocytes and rat liver were prepared by extracting ribosomes with 0.2n-hydrochloric acid or guanidinium chloride and subsequent dialysis. 2. Treatment for 2.5hr. or less with 0.2n-hydrochloric acid dissolved 46-54% of the proteins, which were richer in arginine and lysine and in N-terminal alanine groups and poorer in aspartic acid and glutamic acid and in N-terminal glycine groups than the acid-insoluble proteins. 3. Protein fractions prepared from the guanidinium chloride extract of ribosomes from rat liver were usually more basic than those from rabbit reticulocytes. 4. The ratios lysine: arginine of fractions in the guanidinium chloride extracts were appreciably higher for proteins from rabbit reticulocytes than from rat liver. 5. The concentration of urea and the pH of the gel affected the rate of migration and number of bands in starch-gel electrophoresis.

The metabolic stability of ribosomal protein.

Abstract: Measurements of the metabolic stability of ribosomal proteins of exponentially growing Escherichia coli B/r showed that their rate of degradation to free amino acids is zero to within limits of the experimental accuracy of ± 0.7% per hour or ± 0.5% per doubling period.

Biotin-deficient growth of Bacillus polymyxa.

Abstract: When Bacillus polymyxa, a wild-type biotin auxotroph, is grown in biotin-deficient medium, a retardation of cell division and consequential cell elongation are the initial detectable consequences of limited biotin Subsequent events in biotin-deficient cells include, in chronological order: inhibition of net ribonucleic acid (RNA) synthesis and a simultaneous arithmetical accumulation of protein; loss of net RNA, deoxyribonucleic acid, and protein synthesis; morphological aberration, death, and lysis Incorporation studies employing 32P-phosphate and 14CO2 demonstrate an initial selective inhibition of net ribosomal RNA synthesis over that of ribosomal protein or total protein Biotin could not be replaced by various extracts from which biotin had been removed, nor could osmotic stabilizers be found which could prevent lysis of the culture

Effect of antibiotics on ribosomal RNA and proteins from Streptococcus pyogenes.

Abstract: Cells of Streptococcus pyogenes were prepared under rigid conditions. The microorganisms were then incubated for 3 hours in the presence or absence of chloramphenicol, actinomycin or puromycin. RNA, ribosomal fraction and ribosomal proteins were isolated from the cells. The materials were invesigated with the help of infra red spectroscopy using the potassium bromide pellet method. Quantitative differences in the 1750–1500 cm-1 region were observed with materials treated with the antibiotics. Synthetic mixtures of ribosomal RNA with progressively larger amounts of ribosomal proteins show analogous changes, namely a progressive increase in the strength of the 1650 cm-1 band relative to the 1685 cm-1 band, and an increase in the 1535 cm-1 band. The analytical results obtained with the ribosomal RNA isolated from S. pyogenes treated with antibiotics indicated increased amounts of proteins which could not be removed by the applied extraction method. The evidence presented suggests a change in the binding between ribosomal RNA and ribosomal proteins in the material isolated from the antibiotic treated microorganisms. The I. R. spectroscopy seems to be an useful tool in the investigation of some aspects of biological materials.