scholarly journals Association of nascent polypeptide with 30S ribosomal subunits

1973 ◽  
Vol 136 (4) ◽  
pp. 859-863 ◽  
Author(s):  
Michael Cannon ◽  
M. Amin A. Mirza ◽  
Margaret L. M. Anderson
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Protein Synthesis ◽  
Trichloroacetic Acid ◽  
Sucrose Gradient ◽  
Gradient Centrifugation ◽  
Ribosomal Subunits ◽  
Nascent Polypeptide ◽  
Sucrose Gradient Centrifugation ◽  
Crude Extracts

1. Crude extracts of Escherichia coli were used to synthesize nascent peptides under the direction of endogenous mRNA and in the presence of radioactive amino acids. Analysis of such extracts by sucrose-gradient centrifugation in low Mg2+concentration has shown that after 2min of incubation approximately 14% of the total labelled protein recovered on the gradient, in association with whole ribosomes, sediments with 30S ribosomal subunits; this value rises to approximately 24% after 30min of incubation. The labelled protein associated with 30S ribosomal subunits is insoluble in hot trichloroacetic acid. 2. Similar results were also obtained in extracts that synthesized polypeptides under the direction of either of the synthetic polyribonucleotides poly(A) or poly(A,G,C,U). In contrast, however, analysis of crude extracts programmed in protein synthesis by poly(U) has indicated that under these conditions 30S ribosomal subunits have no associated polyphenylalanine; similarly there is little associated peptide after programming of extracts by poly(U,C).

scholarly journals Association of nascent polypeptide and transfer ribonucleic acid with 30S ribosomal subunits

1973 ◽  
Vol 136 (4) ◽  
pp. 865-869
Author(s):  
M. Amin A. Mirza ◽  
Michael Cannon ◽  
Margaret L. M. Anderson
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Protein Synthesis ◽  
Sucrose Gradient ◽  
Gradient Centrifugation ◽  
Sodium Dodecyl ◽  
Radioactive Material ◽  
Considerable Proportion ◽  
Experimental Conditions ◽  
Ribosomal Subunits

1. Crude extracts of Escherichia coli programmed in protein synthesis by endogenous mRNA have incorporated amino acids into protein. Analysis of such extracts by sucrose-gradient centrifugation in low Mg2+concentration has revealed that 30S ribosomal subunits carry associated radioactive material of which a considerable proportion can be removed from ribosomes by treatment of pre-labelled extracts with puromycin. 2. Gradient analyses of incorporations carried out in the additional presence of added32P-labelled tRNA have indicated that tRNA sediments in the regions of the newly synthesized nascent protein and that both labels are associated with all ribosomal components detected on the gradients under the experimental conditions employed. 3. 30S ribosomal subunits carrying both32P and14C labels have been isolated, disrupted with sodium dodecyl sulphate, and analysed by chromatography on Sephadex G-200 columns. Both labels elute closely together and well away from a tRNA marker analysed under identical conditions. 4. It is proposed that 30S ribosomal subunits, isolated from extracts which have synthesized nascent peptides under the direction of endogenous mRNA, carry associated peptidyl-tRNA.

Structural proteins of a lipid-containing bacteriophage which replicates in Escherichia coli: phage PR4

1977 ◽  
Vol 23 (8) ◽  
pp. 1084-1087 ◽  
Author(s):  
Stephen P. Cadden ◽  
Jeffrey A. Sands
Keyword(s):  
Escherichia Coli ◽  
Viral Protein ◽  
Sucrose Gradient ◽  
Gradient Centrifugation ◽  
Structural Proteins ◽  
Major Protein ◽  
Protein Species ◽  
Sucrose Gradient Centrifugation ◽  
Molecular Weights ◽  
Escherichia Coli Phage

PR4 is a lipid-containing bacteriophage which is able to replicate in Escherichia coli. The virus was labeled with either [14C]leucine and [14C]threonine or H235SO4 and then purified by several rounds of sucrose gradient centrifugation. Autoradiographs showed the virus to be composed of six major protein species with molecular weights (1) 68 000, (2) 47 500, (3) 38 500, (4) 35 000, (5) 20 700, (6) 16 500 daltons. Electropherograms showed protein No. 2 to be the major protein, comprising about 43% of the total weight of viral protein.

Inhibition of the induced synthesis of protocatechuate oxygenase by o-nitrobenzoic acid

1970 ◽  
Vol 16 (7) ◽  
pp. 609-614
Author(s):  
Karen F. Montgomery ◽  
Norman N. Durham
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Protein Synthesis ◽  
Trichloroacetic Acid ◽  
Protocatechuic Acid ◽  
Viable Cell ◽  
Insoluble Fraction ◽  
Nitrobenzoic Acid ◽  
Cell Counts ◽  
Oxygenase Activity

The synthesis of protocatechuate oxygenase (EC. 1.13.1.3), an inducible enzyme, by Pseudomonas fluorescens was inhibited by o-nitrobenzoic acid. The inhibitor did not alter the oxygenase activity of induced cells. Viable cell counts indicated that o-nitrobenzoic acid, in the concentrations used in these experiments, did not kill the cells. The inhibitor decreased the incorporation of L-tryptophan-14C and DL-glutamic-14C acid into the hot trichloroacetic acid insoluble fraction of the cell, but had no effect on the uptake of either radioactive amino acids or the inducer, protocatechuic acid. The synthesis of β-galactosidase by Escherichia coli was also inhibited by o-nitrobenzoic acid. The results establish that one site of o-nitrobenzoic acid inhibition is associated with protein synthesis in the cell.

ANALYSIS OF RADIOIMMUNOASSAY BY DISC ELECTROPHORESIS AND SUCROSE GRADIENT CENTRIFUGATION

1971 ◽  
Vol 66 (2) ◽  
pp. 257-265 ◽  
Author(s):  
Lubomir Valenta ◽  
Michel L. Aubert
Keyword(s):  
Sucrose Gradient ◽  
Solid Phase ◽  
Gradient Centrifugation ◽  
Human Growth ◽  
Disc Electrophoresis ◽  
Separate Analysis ◽  
Adrenocorticotrophic Hormone ◽  
Sucrose Gradient Centrifugation ◽  
Before And After ◽  
Polypeptide Hormones

ABSTRACT Radioiodine-labelled synthetic adrenocorticotrophic hormone (ACTH), human growth hormone (HGH), human chorionic somato-mammotrophin (HCS), and human (HTSH) and bovine (BTSH) thyroid stimulating hormones were studied by disc-electrophoresis and sucrose gradient centrifugation before and after incubation with corresponding antisera. All antisera contained 7 S antibodies. After incubation, soluble antigenantibody complexes besides a small amount of precipitate were observed in the incubation mixture, characteristic of each hormone. The complexes migrated like gamma globulins or more slowly on disc-electrophoresis. and on sucrose gradient centrifugation showed patterns dependent on the time of incubation. Light 7 or 9 S, or < 12 S complexes occurred mostly after incubation for several minutes (up to 30 min) before analysis. When incubation was prolonged to 24 h and more, these relatively light complexes disappeared or diminished in favour of heavier soluble or precipitating complexes. Reproducibly obtainable sedimentation patterns of the soluble complexes suggested some definite recombination of antigen molecules with 7 S antibodies. The complexes did not occur on incubation with other sera than an antiserum to a given hormone. They were not influenced by EDTA. Displacement of the radioactivity of the complexes into the free hormone peak was obtained by addition of a non-labelled hormone identical with the labelled one. Sucrose gradient centrifugation and disc-electrophoresis are recommended for the study of immunoreaction of diluted materials and for a separate analysis of different steps of the radioimmunoassay. Radioimmunoassay was introduced for the measurement of protein hormones by Yalow & Berson (1960). The method, described originally for insulin, was later adapted to the detection of a number of protein and polypeptide hormones. On incubation of the hormone with its antiserum, a soluble antigenantibody complex is formed, which is separated from an excess of the free hormone by various methods, e. g. chromatoelectrophoresis, precipitation with a second antibody, adsorption on a solid phase etc. (Hunter 1967). Sucrose gradient centrifugation and disc-electrophoresis were occasionally used to follow some isolated aspect of radioimmunoassay (Fitschen 1965; Monjardino et al. 1968). We are demonstrating that these methods made it possible to analyze the radioimmunoassay step by step and thus may be useful for practical purposes as well as in a study of the immunoreaction of diluted materials.

scholarly journals LOCALIZED MUTAGENESIS FOR THE ISOLATION OF TEMPERATURE-SENSITIVE MUTANTS OF ESCHERICHIA COLI AFFECTED IN PROTEIN SYNTHESIS

Genetics ◽  
1979 ◽  
Vol 91 (2) ◽  
pp. 215-227
Author(s):  
W Scott Champney
Keyword(s):  
Escherichia Coli ◽  
Protein Synthesis ◽  
Temperature Sensitive ◽  
Chromosomal Locus ◽  
Ribosomal Subunits ◽  
Prolonged Incubation ◽  
Temperature Sensitive Mutants ◽  
Inhibition Of Growth ◽  
Localized Mutagenesis

ABSTRACT Two variations of the method of localized mutagenesis were used to introduce mutations into the 72 min region of the Escherichia coli chromosome. Twenty temperature-sensitive mutants, with linkage to markers in this region, have been examined. Each strain showed an inhibition of growth in liquid medium at 44°, and 19 of the mutants lost viability upon prolonged incubation at this temperature. A reduction in the rate of in vivo RNA and protein synthesis was observed for each mutant at 44°, relative to a control strain. Eleven of the mutants were altered in growth sensitivity or resistance to one or more of three ribosomal antibiotics. The incomplete assembly of ribosomal subunits was detected in nine strains grown at 44°. The characteristics of these mutants suggest that many of them are altered in genes for translational or transcriptional components, consistent with the clustering of these genes at this chromosomal locus.

scholarly journals LYSOSOMAL PHOSPHOLIPASES A1 AND A2 OF NORMAL AND BACILLUS CALMETTE GUERIN-INDUCED ALVEOLAR MACROPHAGES

1973 ◽  
Vol 56 (3) ◽  
pp. 621-627 ◽  
Author(s):  
Richard C. Franson ◽  
Moseley Waite
Keyword(s):  
Acid Phosphatase ◽  
Alveolar Macrophages ◽  
Sucrose Gradient ◽  
Gradient Centrifugation ◽  
Marker Enzymes ◽  
Sucrose Gradient Centrifugation ◽  
Control Distribution ◽  
Specific Activities ◽  
Selective Increase ◽  
Phospholipases A

A single intravenous injection of 0.1 mg of heat-killed Bacillus Calmette Guérin (BCG) in 0.1 ml of Bayol F produced an accumulation of activated alveolar macrophages (BCG induced). Cells were collected 3.5–4.0 wk after injection. Phospholipases A and three lysosomal marker enzymes (acid phosphatase, ß-glucuronidase, and lysozyme) were measured in homogenates, and the distribution of the phospholipases A and lysosomal, mitochondrial, and microsomal marker enzymes were examined after sucrose gradient centrifugation of a postnuclear (1,000 g) supernatant. Homogenates of normal and BCG-induced macrophages contained phospholipases A1 and A2 which had optimal activity at pH 4.0 in the presence of 2.0 mM ethylenediaminetetraacetate (EDTA). These activities were inhibited 50–70% by 2.0 mM CaCl2. Homogenates of BCG-induced macrophages had specific activities of ß-glucuronidase, acid phosphatase, and lysozyme, which were increased 1.5- to 3.0-fold over the controls, whether expressed as activity per mg protein or activity per 107 cells. The specific activities of the phospholipases A, on the other hand, were consistently lower than those of the control. Distribution of the phospholipases A and the lysosomal marker enzymes after sucrose gradient centrifugation suggested that the phospholipases A active at pH 4.0 in the presence of EDTA are of lysosomal origin since: (a) BCG treatment caused a selective increase in the density of particles which contained both the phospholipases A and three lysosomal marker enzymes; and (b) since the density of mitochondria and microsomes were not affected by BCG treatment. The increase in the density of lysosomes seen here may be related to previously described morphologic changes of BCG-induced alveolar macrophages.

scholarly journals Degradation of surfactant-associated protein B (SP-B) during in vitro conversion of large to small surfactant aggregates

1993 ◽  
Vol 295 (1) ◽  
pp. 141-147 ◽  
Author(s):  
R A W Veldhuizen ◽  
K Inchley ◽  
S A Hearn ◽  
J F Lewis ◽  
F Possmayer
Keyword(s):  
Surface Area ◽  
Sucrose Gradient ◽  
Gradient Centrifugation ◽  
Sucrose Gradient Centrifugation ◽  
Aggregate Fraction ◽  
Surfactant Aggregates ◽  
Morphological Studies ◽  
Surface Active ◽  
Protein B

Pulmonary surfactant obtained from lung lavages can be separated by differential centrifugation into two distinct subfractions known as large surfactant aggregates and small surfactant aggregates. The large-aggregate fraction is the precursor of the small-aggregate fraction. The ratio of the small non-surface-active to large surface-active surfactant aggregates increases after birth and in several types of lung injury. We have utilized an in vitro system, surface area cycling, to study the conversion of large into small aggregates. Small aggregates generated by surface area cycling were separated from large aggregates by centrifugation at 40,000 g for 15 min rather than by the normal sucrose gradient centrifugation. This new separation method was validated by morphological studies. Surface-tension-reducing activity of total surfactant extracts, as measured with a pulsating-bubble surfactometer, was impaired after surface area cycling. This impairment was related to the generation of small aggregates. Immunoblot analysis of large and small aggregates separated by sucrose gradient centrifugation revealed the presence of detectable amounts of surfactant-associated protein B (SP-B) in large aggregates but not in small aggregates. SP-A was detectable in both large and small aggregates. PAGE of cycled and non-cycled surfactant showed a reduction in SP-B after surface area cycling. We conclude that SP-B is degraded during the formation of small aggregates in vitro and that a change in surface area appears to be necessary for exposing SP-B to protease activity.

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