Protein synthesis by cell-free extracts of Proteus vulgaris

1971 ◽  
Vol 17 (12) ◽  
pp. 1545-1551
Author(s):  
K. L. Backler ◽  
W. E. Inniss
Keyword(s):  
Protein Synthesis ◽  
Ribonucleic Acid ◽  
Supernatant Fraction ◽  
Proteus Vulgaris ◽  
Guanosine Triphosphate ◽  
Cell Extracts ◽  
Polyuridylic Acid ◽  
A Cell ◽  
Directed System ◽  
Low Activity

A cell-free protein-synthesizing system from Proteus vulgaris was developed and characterized. Initially, a very low activity was found to occur; it was shown to be due to a deficiency in the endogenous transfer ribonucleic acid (tRNA) content of the cell extracts. When tRNA was added to the polyuridylic acid (poly U) directed system, incorporation of 14C-phenylalanine into polypeptide readily occurred. This incorporation was dependent on ribosomes and supernatant fraction and the optimum concentrations of the various components of the system were found to be as follows: 120 μg/ml tRNA, 30 mM magnesium, 0.5 mM spermidine, 60 mM potassium, 50 mM adenosine triphosphate (ATP), 5 mM guanosine triphosphate (GTP), 0.03 mM cytosine triphosphate (CTP) and uridine triphosphate (UTP), 5 mM phosphoenolpyruvate (PEP), 50 μg/ml poly U, and 30 mM 2-mercaptoethanol.

Proteinsynthese mit Zellextrakten von Micrococcus radiodurans/ Protein Synthesis with Cell Extracts of Micrococcus radiodurans

1978 ◽  
Vol 33 (11-12) ◽  
pp. 948-954
Author(s):  
Annette Widmann ◽  
Roland Süssmuth
Keyword(s):  
Protein Synthesis ◽  
Optimal Conditions ◽  
Free System ◽  
Cell Free System ◽  
E Coli ◽  
Cell Extracts ◽  
Polyuridylic Acid ◽  
A Cell ◽  
Ribosomal Activity ◽  
Cell Free Protein Synthesis

Abstract Pure active ribosomes of cells of Micrococcus radiodurans could be obtained when cultivated in trypton, glucose and nutrient broth by adding natrium citrate. The optimal conditions for a cell-free protein synthesis were investigated at the (polyuridylic acid) dependent polyphenylalanine synthesis. When exchanging ribosomes and S100-fractions with the corresponding fractions of E. coli, we found that the enzyme fractions of M. radiodurans extremely inhibit the ribosomal activity. The incorporation rates in the cell-free system of M. radiodurans yield, at com parable conditions, in relation to E. coli under 10%.

scholarly journals Protein synthesis in the cotyledons of Pisum sativum L. Protein factors involved in the binding of phenylalanyl-transfer ribonucleic acid to ribosomes*

1974 ◽  
Vol 139 (1) ◽  
pp. 61-69 ◽  
Author(s):  
Gary N. Wells ◽  
Leonard Beevers
Keyword(s):  
Protein Synthesis ◽  
Complex Formation ◽  
Pisum Sativum ◽  
Ribonucleic Acid ◽  
Ternary Complex ◽  
Supernatant Fraction ◽  
Soluble Factors ◽  
Pisum Sativum L ◽  
Sensitive Site ◽  
Millipore Filters

1. Proteinaceous factors contained in a 0.5m-KCl extract of ribosomes from pea cotyledons form a ternary complex at 0°C with [14C]phenylalanyl-tRNA and poly(U). The complex is measured by its quantitative retention on Millipore filters. 2. Complex-assembly is optimal at 5mm-Mg2+ and is independent of GTP and ribosomes. 3. The addition of ribosomes is required to stabilize the complex at 34°C. The complex binds to a puromycin-sensitive site on the ribosome. 4. Soluble factors from the 250000g supernatant of pea cotyledon form a Millipore-retainable complex dependent on GTP and ribosomes. 5. Complex-formation by soluble factors has a Mg2+ optimum of 10–12mm and forms a puromycin-insensitive complex with ribosomes. 6. The function of the ribosomal protein factors and the supernatant fraction in initiation of protein synthesis is discussed.

Nucleo-cytoplasmic interactions in the regulation of thymidine phosphorylation in Acetabularia

1977 ◽  
Vol 198 (1131) ◽  
pp. 177-190 ◽  
Keyword(s):  
Protein Synthesis ◽  
Enzyme Activity ◽  
Cell Nucleus ◽  
Kinase Activity ◽  
De Novo ◽  
Maximum Diameter ◽  
Enzyme Protein ◽  
Activity Increase ◽  
Cell Extracts ◽  
Low Activity

The activity of dT kinase (EC. 2. 7. 1. 75) was estimated during the development of nucleate and anucleate cells in Acetabularia . Enzyme activity increases as the cap attains its maximum diameter (Bannwarth & Schweiger 1975). Regulation of the dT kinase activity is observed even in the absence of the cell nucleus. Twelve to fifteen days after enucleation dT kinase activity increase sharply as the cap reaches its maximum diameter and decreases 5 days later. The increase is inhibited by puromycin, chloramphenicol, rifampicin and ethidium bromide but not by cycloheximide. These findings indicate that protein synthesis on 70S organelle ribosomes is involved in the regulation of the dT kinase. Since activities of mixtures from low activity cell extracts and from high activity cell extracts are additive, it is concluded that the regulation is not mediated through an activator protein but rather through de novo synthesis of the enzyme protein.

Action of streptomycin on the growth of Streptomyces griseus

1974 ◽  
Vol 20 (11) ◽  
pp. 1591-1597 ◽  
Author(s):  
R. Cella ◽  
L. C. Vining
Keyword(s):  
Protein Synthesis ◽  
Natural Population ◽  
Streptomyces Griseus ◽  
Drastic Reduction ◽  
Cell Extracts ◽  
Polyuridylic Acid ◽  
Directed Polymerization ◽  
Selection Of

A streptomycin-producing culture of Streptomyces griseus was sensitive to streptomycin, but inhibition was temporary, and cultures supplemented with the antibiotic up to 100 μg/ml grew after a lag. Streptomycin tolerance developed, not by inactivation of the antibiotic but by selection of resistant variants in the natural population. Addition of streptomycin to growing cultures caused a drastic reduction in protein synthesis and an accumulation of "stuck" (70 S) ribosomes within the mycelium. Although the effect on protein synthesis could not be confirmed in vitro because cell extracts from S. griseus contained an inhibitor of polyuridylic-acid-directed polymerization of L-phenylalanine, it is concluded that streptomycin prevents the growth of this organism by a mechanism similar to that observed with other bacteria and that the tolerance of producing cultures cannot be attributed to the presence of streptomycin-resistant ribosomes.

scholarly journals Cell-free synthesis of tryptophanase from Escherichia coli. Use of ribonucleic acid isolated from induced cells and a comparison of the product from a system employing ribosomes with that from one employing ribosomes and exogenous ribonucleic acid

1971 ◽  
Vol 125 (2) ◽  
pp. 643-653 ◽  
Author(s):  
J. H. Parish ◽  
S. A. M. Khairul Bashar ◽  
N. L. Brown ◽  
Marjorie Brown
Keyword(s):  
Protein Synthesis ◽  
Amino Acid ◽  
Ribonucleic Acid ◽  
Supernatant Fraction ◽  
23S Rrna ◽  
Terminal Amino Acid ◽  
Tryptic Peptides ◽  
Radioactive Amino Acid ◽  
Terminal Amino

1. Polyribosomes and RNA were isolated from cultures in which tryptophanase (EC 4.2.1.–) was induced. The polyribosomes were incubated under conditions of protein synthesis, in the presence of a radioactive amino acid and a post-ribosomal supernatant fraction obtained from repressed cells. The RNA preparations were incubated under conditions of protein synthesis in the presence of a radioactive amino acid and a supernatant fraction containing ribosomes from repressed cells. 2. The system was characterized and the synthesis of a radioactive protein with the same chromatographic properties as tryptophanase was demonstrated. This synthesis was shown to be time-dependent and required the presence of RNA from induced cultures, ribosomes and an energy supply; it was inhibited by chloramphenicol. 3. The maximum activity for the synthesis of this protein was found to be associated with 23S rRNA isolated from sucrose gradients. 4. The N-terminal amino acid of tryptophanase was labelled in the protein synthesized in this system but not in the protein synthesized by polyribosomes (without added RNA). Conversely, the C-terminal amino acid of tryptophanase was labelled in the polyribosome system but not in the RNA-containing system. 5. Tryptic digests of protein labelled in vitro were compared with those of tryptophanase. No labelled tryptic peptides were identified other than tryptophanase tryptic peptides. An analysis of the results implied that in the polyribosome system almost the complete tryptophanase subunit chain was labelled but that in the RNA-containing system these chains were incompletely synthesized. 6. Sucrose-gradient analysis of protein synthesized in the RNA-containing system suggested that it cannot be converted into structures with the same sedimentation properties as native tryptophanase. 7. The significance of these results for the assay of tryptophanase mRNA and for an understanding of the control of the translation of this mRNA in vivo is discussed.

Mechanism of action of an inhibitor from Proteus vulgaris on cell-free protein synthesis by Escherichia coli B

1969 ◽  
Vol 15 (2) ◽  
pp. 159-164
Author(s):  
J. J. McEvoy ◽  
W. E. Inniss
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Protein Synthesis ◽  
Mechanism Of Action ◽  
Ribonucleic Acid ◽  
Proteus Vulgaris ◽  
Flash Evaporation ◽  
Free System ◽  
Messenger Ribonucleic Acid ◽  
Escherichia Coli B

An inhibitory substance(s) has been found in S-30 fractions from Proteus vulgaris which prevented an Escherichia coli B cell-free system from incorporating a mixture of 14C-amino acids, L-phenylalanine-14C, or L-lysine-14C into protein, as directed by natural messenger ribonucleic acid, polyuridylic acid, or polyadenylic acid respectively. Similar results were obtained when the inhibitor was isolated from S-100 fractions by using dialysis, concentration of the dialysate by flash evaporation, hydrolysis, evaporation to dryness, dissolution to the original volume in distilled water, and neutralization. The effect of the inhibitor on the various individual reactions involved in protein synthesis was examined. No effect was found on the activation of amino acids as determined by the formation of L-phenylalanine-14C hydroxamate isolated chromatographically or by adenosine triphosphate – pyrophosphate exchange. Also no inhibition of L-phenylalanine-14C attachment to transfer ribonucleic acid occurred. However, ribosome-dependent reactions were markedly inhibited. The mechanism of action of the inhibitor appeared to be the prevention of binding of phenylalanyl-transfer ribonucleic acid to the ribosomes.

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