Studies on the Incorporation of Amino Acids by a Cell-Free System Obtained from Beef Retina

1972 ◽  
Vol 50 (5) ◽  
pp. 581-587 ◽  
Author(s):  
Y. Matuk
Keyword(s):  
Amino Acids ◽  
Protein Synthesis ◽  
Incubation Medium ◽  
Optimum Concentration ◽  
Free System ◽  
Cell Free System ◽  
A Cell

The incorporation of 14C-leucine into proteins by a cell-free system from beef retina was studied. It was found that the optimum concentration of ATP depended on the concentration of ribosomes in the incubation medium. Very little incorporation of 14C-leucine was observed in the absence of K+. The optimum concentration of phosphocreatine required for incorporation of radioactive leucine depended on the concentration of Mg2+ in the incubation medium, and the optimum concentration of K+ appears to be independent of the concentrations of Mg2+ and phosphocreatine used.Retinol and retinal had no effect, but ethanol markedly inhibited protein synthesis at concentrations higher than 2%.Puromycin (10−4 M) inhibited incorporation of 14C-leucine by about 80%. The degree of inhibition by cycloheximide depended on the concentration of pH 5 fraction in the incubation medium.

scholarly journals Inhibition of protein synthesis in Krebs 2 ascites cells and cell-free systems by phenylalanine and its effect on leucine and lysine in the amino acid pool

1968 ◽  
Vol 109 (4) ◽  
pp. 507-515 ◽  
Author(s):  
Joan P. Roscoe ◽  
M. D. Eaton ◽  
Gladys Chin Choy
Keyword(s):  
Amino Acids ◽  
Protein Synthesis ◽  
Specific Radioactivity ◽  
Transfer Rna ◽  
Free System ◽  
Cell Free System ◽  
Whole Cells ◽  
System L ◽  
Inhibition Of Protein Synthesis ◽  
A Cell

1. The inhibition of incorporation of 14C-labelled amino acids into protein of whole cells by phenylalanine has been reproduced in a cell-free system. In both cases only the l-isomer was inhibitory. 2. The effect of phenylalanine on incorporation of [14C]leucine and [14C]lysine into protein was different in both whole cells and cell-free systems. 3. In whole cells inhibition of incorporation of leucine at 2·5μg./ml. was very rapid, but when the concentration was increased to 100μg./ml. the inhibition was not apparent for about 1hr. The kinetics of inhibition of lysine was the same at both these concentrations and was similar to that found with leucine at 100μg./ml. 4. Neither a lower specific radioactivity of the two amino acids in the pool nor a decrease in their pool size could be consistently related with inhibition of protein synthesis. 5. In the cell-free system l-phenylalanine inhibited the incorporation of leucine but not of lysine. 6. Charging of transfer RNA by leucine was markedly decreased in the presence of phenylalanine, whereas charging of transfer RNA by lysine was not.

Initiation in einem Polyribosomen-abhängigen Protein-synthetisierenden zellfreien System aus Saccharomyces / Initiation in a Polyribosome-Dependent Protein-Synthesizing Cell-Free System from Saccharomyces

1976 ◽  
Vol 31 (3-4) ◽  
pp. 169-173 ◽  
Author(s):  
Bernd Schulz-Harder ◽  
Ernst-Randolf Lochmann
Keyword(s):  
Protein Synthesis ◽  
Free System ◽  
Cell Free System ◽  
Aurintricarboxylic Acid ◽  
Dependent Protein ◽  
A Cell

Abstract A method to prepare polyribosomes from yeasts by using the french-press is described. The highest yield of polyribosomes was derived from late log-phase cells. These polyribosomes, incubated in a cell-free system, were able to reinitiate protein synthesis, which was shown by inhibiting aminoacid incorporation by aurintricarboxylic acid, edeine and sodiumfluoride. We developed the translational system in order to look for the optimal ion-conditions of a DNA-dependent protein-synthesizing system. We found out that at the optimal MgCL2-concentration (6 mᴍ) protein synthesis was strongly inhibited by Mangan ions which are required for transcription in yeast. If protein-synthesis was carried out with 2 mᴍ and 3 mᴍ MgCl2 maximal aminoacid incorporation was observed at 2 mᴍ and 1.5 mᴍ MnCl2.

scholarly journals Cell-Free Protein Synthesis by Diversifying Bacterial Transcription Machinery

BioTech ◽  
2021 ◽  
Vol 10 (4) ◽  
pp. 24
Author(s):  
Marina Snapyan ◽  
Sylvain Robin ◽  
Garabet Yeretssian ◽  
Michèle Lecocq ◽  
Frédéric Marc ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Genomic Sequence ◽  
Synthetic Activity ◽  
Translation System ◽  
Free System ◽  
Transcription Terminator ◽  
Cell Free System ◽  
E Coli ◽  
Bacterial Transcription ◽  
A Cell

We have evaluated several approaches to increase protein synthesis in a cell-free coupled bacterial transcription and translation system. A strong pargC promoter, originally isolated from a moderate thermophilic bacterium Geobacillus stearothermophilus, was used to improve the performance of a cell-free system in extracts of Escherichia coli BL21 (DE3). A stimulating effect on protein synthesis was detected with extracts prepared from recombinant cells, in which the E. coli RNA polymerase subunits α, β, β’ and ω are simultaneously coexpressed. Appending a 3′ UTR genomic sequence and a T7 transcription terminator to the protein-coding region also improves the synthetic activity of some genes from linear DNA. The E. coli BL21 (DE3) rna::Tn10 mutant deficient in a periplasmic RNase I was constructed. The mutant cell-free extract increases by up to four-fold the expression of bacterial and human genes mediated from both bacterial pargC and phage pT7 promoters. By contrast, the RNase E deficiency does not affect the cell-free expression of the same genes. The regulatory proteins of the extremophilic bacterium Thermotoga, synthesized in a cell-free system, can provide the binding capacity to target DNA regions. The advantageous characteristics of cell-free systems described open attractive opportunities for high-throughput screening assays.

scholarly journals The Capacity of Polyadenylated RNA from Myogenic Cells Treated with Actinomycin D to Direct Protein Synthesis in a Cell-Free System

1978 ◽  
Vol 88 (2) ◽  
pp. 403-410 ◽  
Author(s):  
Gania KESSLER-ICEKSON ◽  
Robert H. SINGER ◽  
David YAFFE
Keyword(s):  
Protein Synthesis ◽  
Actinomycin D ◽  
Myogenic Cells ◽  
Free System ◽  
Cell Free System ◽  
Polyadenylated Rna ◽  
A Cell

Isolation and partial characterization of amphibian tyrosine oxidase polysomes

Development ◽  
1970 ◽  
Vol 24 (1) ◽  
pp. 109-118
Author(s):  
E. L. Triplett ◽  
R. Herzog ◽  
L. P. Russell
Keyword(s):  
Amino Acids ◽  
Protein Synthesis ◽  
Amino Acid ◽  
Enzymic Activity ◽  
Antigenic Determinants ◽  
Free System ◽  
A Cell ◽  
Generating System ◽  
Soluble Components

A population of polysomes isolated from frogskinis capable of supporting protein synthesis in a cell-free system containing an energy generating system, ‘soluble components’, and amino acids. These polysomes catalyse the oxidation of DOPA after gentle trypsinization, and they also have antigenic determinants attributable to tyrosine oxidase. Skin polysomes sedimented in 10–30 % sucrose gradients contain tyrosine oxidase peaks of enzymic activity at the bottom and top of the tube and in the 250 S regions. A peak of tyrosine oxidase antigenic acitvity is found in the 250–350S region of the gradient. Polysomes resolved on the gradient retain the ability to support protein synthesis in a cellfree system. All 250–350S particles capable of supporting the incorporation of [14C]amino acid into tyrosine oxidase are precipitable with tyrosine oxidase antibodies. It is probable that 250–350S tyrosine oxidase antibody precipitates contain only polysomes for this protein.

scholarly journals Protein synthesis in a cell-free system coded by poly(A)-mRNA isolated from bovine retina

1981 ◽  
Vol 31 ◽  
pp. 98
Author(s):  
Che-Hui Kuo ◽  
Naomasa Miki
Keyword(s):  
Protein Synthesis ◽  
Free System ◽  
Cell Free System ◽  
Bovine Retina ◽  
A Cell

Initiation of protein synthesis in a cell-free system prepared from rat hepatocytes

1989 ◽  
Vol 256 (1) ◽  
pp. C28-C34 ◽  
Author(s):  
S. R. Kimball ◽  
W. V. Everson ◽  
K. E. Flaim ◽  
L. S. Jefferson
Keyword(s):  
Protein Synthesis ◽  
Amino Acid ◽  
Rat Hepatocytes ◽  
Initiation Factor ◽  
Nucleotide Exchange ◽  
Isolated Rat Hepatocytes ◽  
Intact Cells ◽  
Free System ◽  
Cell Free System ◽  
A Cell

A cell-free system, which maintained a linear rate of protein synthesis for up to 20 min of incubation, was prepared from isolated rat hepatocytes. The rate of protein synthesis in the cell-free system was approximately 20% of the rate obtained in isolated hepatocytes or perfused liver. More than 70% of total protein synthesis in the cell-free system was due to reinitiation, as indicated by addition of inhibitors of initiation, i.e., edeine or polyvinyl sulfate. The rate of protein synthesis and formation of 43S initiation complexes in the cell-free system were reduced to 60 and 30% of the control values, respectively, after incubation of hepatocytes in medium deprived of an essential amino acid. Therefore, the cell-free system maintained the defect in initiation induced in the intact cells by amino acid deprivation. The defect in initiation was corrected by addition of either rat liver eukaryotic initiation factor 2 or the guanine nucleotide exchange factor (GEF) to the cell-free system. A role for GEF in the defect in initiation was further implicated by experiments that showed that the activity of the factor was decreased in extracts from livers perfused with medium deficient in amino acids. The cell-free system should provide a valuable tool for investigation of mechanisms involved in the regulation of initiation of protein synthesis.

scholarly journals Structure-activity Relationships of Hygromycin A and Its Analogs: Protein Synthesis Inhibition Activity in a Cell Free System.

1997 ◽  
Vol 50 (6) ◽  
pp. 514-521 ◽  
Author(s):  
SHIGERU F. HAYASHI ◽  
LAURA J. L. NORCIA ◽  
SCOTT B. SEIBEL ◽  
ANNETTE M. SILVIA
Keyword(s):  
Protein Synthesis ◽  
Protein Synthesis Inhibition ◽  
Inhibition Activity ◽  
Free System ◽  
Synthesis Inhibition ◽  
Cell Free System ◽  
Structure Activity Relationships ◽  
Structure Activity ◽  
A Cell

Incorporation of 14C-Amino Acids into Basic Proteins of 47S and 32S Subunits of Rat Liver Ribosomes by a Cell-Free System

1968 ◽  
Vol 64 (3) ◽  
pp. 407-409 ◽  
Author(s):  
KAZUO TERAO ◽  
HIROSHI SUGANO ◽  
KIKUO OGATA
Keyword(s):  
Amino Acids ◽  
Rat Liver ◽  
Free System ◽  
Basic Proteins ◽  
Cell Free System ◽  
A Cell ◽  
Liver Ribosomes
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