Mode of action of chloramphenicol IX. Effects of chloramphenicol upon a ribosomal amino acid polymerization system and its binding to bacterial ribosome

Wolfe, Alan D.; Hahn, Fred E.

doi:10.1016/0005-2787(65)90219-4

Cited by 131 publications

(42 citation statements)

References 11 publications

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“…Chloramphenicol inhibits in vivo protein biosynthesis completely (7). However, the inhibitory effect of chloramphenicol is not so strong in the poly(U)-directed in vitro protein-synthesizing system (15). We have found that chloramphenicol inhibits polyphenylalanine synthesis in vitro by about 50% (Table 1), which is in agreement with published results (15,16).…”

Section: Methodssupporting

confidence: 92%

“…However, the inhibitory effect of chloramphenicol is not so strong in the poly(U)-directed in vitro protein-synthesizing system (15). We have found that chloramphenicol inhibits polyphenylalanine synthesis in vitro by about 50% (Table 1), which is in agreement with published results (15,16). A similar degree of inhibition was observed when ribosomes of E. coli and of B. stearothermophilus were incubated at 370 with monobromamphenicol or monoiodoamphenicol instead of chloramphenicol.…”

Section: Methodssupporting

confidence: 92%

“…Then the subunits were spun down by high-speed centrifugation (50 Ti; 40,000 rpm; 6 hr). Ribosomal proteins were extracted and separated as described (15). Each spot, which contained ribosomal protein, was cut into 0.15-cm3 aliquots and dissolved overnight in 1 ml soluene TM (Packard, Lot No.…”

Section: Methodsmentioning

confidence: 99%

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Identification of Chloramphenicol-Binding Protein in Escherichia coli Ribosomes by Affinity Labeling

Pongs

Bald

Erdmann

1973

Proc. Natl. Acad. Sci. U.S.A.

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Monoiodoamphenicol, a synthetic analogue of chloramphenicol, has been shown by competition experiments with chloramphenicol and lincomycin to bind at the same site of 70S ribosomes as chloramphenicol. At -2°it forms a 1:1 complex with 70S ribosomes having a value of K (7.5 X 104 M-1) that is one order of magnitude lower than that of chloramphenicol. At 370, monoiodoamphenicol irreversibly inhibits the protein-synthesizing activity of E. coli ribosomes. It is shown that the analogue reacted preferentially with protein L16 of E. coli 70S ribosomes, and we therefore conclude that protein L16 belongs to the chloramphenicol-binding site of E. coli ribosomes.Since the chemically reactive group of monoiodoamphenicol resembles iodoacetamide, the reaction of E. coli 70S ribosomes with monoiodoamphenicol was compared to that with iodoacetamide. Iodoacetamide did not react with protein L16, but it predominantly reacted with proteins S18 and S21 of the 30S subunit. Furthermore, monoiodoamphenicol was reacted with E. coli ribosomal subunits. Isolated 50S subunits bound monoiodoamphenicol by about one order of magnitude less than 70S ribosomes. Again, protein L16 reacted with the affinity label. Monoiodoamphenicol reacted with protein S18 in isolated 30S subunits; it also bound to 70S ribosomes of Bacillus stearothermophilus, however, it did not bind irreversibly to these 70S ribosomes.A detailed understanding of structure-function relationship of the ribosome is not available (for review see ref. 1). The many elegant approaches that have been tried towards this goal have shown how difficult it is to assign unanimously specific functions to individual ribosomal proteins. Studies of mutants of ribosomal proteins (2), reconstitution of ribosomal particles missing one protein (3), and stimulation of ribosomal functions by addition of ribosomal proteins (4) have shed some light on the great complexity of the ribosomal particle. A promising approach for testing the function of individual ribosomal proteins is the technique of affinity labeling. This technique allows the direct identification of the specific function of an individual ribosomal protein.Recently, the covalent attachment of peptidyl-tRNA analogues to Escherichia coli ribosomes was reported (5, 6).These experiments open the possibility to identify some of the ribosomal proteins involved in binding of peptidyl-tRNA. This report presents for the first time results of studies that probe chemically the chloramphenicol-binding region of E. coli ribosomes by reacting ribosomes with chloramphenicol analoguest. Chloramphenicol has been known for many years as a strong but reversible inhibitor of protein biosynthesis (7), which probably acts at or near the peptidyl-transferase center of the ribosome (8). Chloramphenicol was modified such that it did not lose its antibiotic specificity. The chemically reactive group introduced into the antibiotic was expected to react preferentially and irreversibly with a properly oriented amino-acid functional group in the binding regi...

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Section: Methodssupporting

confidence: 92%

Section: Methodssupporting

confidence: 92%

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Identification of Chloramphenicol-Binding Protein in Escherichia coli Ribosomes by Affinity Labeling

Pongs

Bald

Erdmann

1973

Proc. Natl. Acad. Sci. U.S.A.

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“…Similar phenomena which suggest certain functional relationships between chloramphenicol and erythromycin binding sites has been reported (18,27,29,30,36,40). It has been reported and summarized in several studies that ribosomes often show alteration in their response to antibiotics when selection has been carried out by unrelated antibiotics (1, 2, 4, 21).…”

Section: Downloaded Fromsupporting

confidence: 76%

Pleiotropic Antibiotic Resistance Mutations Associated with Ribosomes and Ribosomal Subunits in Mycobacterium smegmatis

Yamada

Masuda

Shoji

et al. 1974

Antimicrob Agents Chemother

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Viomycin-resistant strains isolated from Mycobacterium smegmatis demonstrated pleiotropic resistance to tuberactinomycin-N, capreomycin, streptomycin, and kanamycin as a result of mutational alteration of ribosomes, even though they were selected for resistance to a single antibiotic. The pleiotropic drug resistance of three mutants isolated by stepwise selection for resistance to viomycin was due to alteration of the 30S ribosomal subunit. One mutant, strain A, isolated independently by multiple-step selection to viomycin resistance, was resistant to viomycin, tuberactinomycin-N, and capreomycin through an alteration of the 50S ribosomal subunit, whereas it was sensitive to kanamycin but resistant to streptomycin through an alteration of the 30S ribosomal subunit. Three streptomycin-resistant strains, which were isolated by one-step selection at a high concentration of streptomycin, did not show significant co-resistance to any other antibiotics tested in culture and cell-free systems; streptomycin resistance in these mutants was localized on the 30S ribosomal subunit.Pleiotropic drug resistance associated with viomycin (VM) resistance in mycobacteria has been reported (6,9,12,13,14,15,25,26,31,32,33,34,37,38,39). Three possible mechanisms for this drug resistance might be considered: (i) enzymatic inactivation of the drugs (17), (ii) alteration of the membrane leading to a change in permeability, and (iii) alterations at the site of action of the antibiotics, e.g., ribosomes (44).As a continuation of our work, pleiotropic drug resistance was studied in culture and in cell-free systems. The results indicate that mutational alteration of the VM binding site on ribosomes led to pleiotropic drug resistance in Mycobacterium smegmatis.The results were presented in abstract form at the XXIInd International Tuberculosis Conference (T. Yamada et al., XXIInd Int. Tuberc.

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“…The results of the present study show that accumulation of chloramphenicol after 5 min by sensitive strains of E. coli and P. aeruginosa reaches respectively about 140 and 97 times the concentration in the medium. When the amount of ribosome-bound chloramphenicol, calculated on the basis of 26,000 ribosomes per cell (Goldstein & Lowney, 1964) and one specific binding site per ribosome (Wolfe & Hahn, 1965;Das, Goldstein & Kenner, 1966;Hurwitz & Braun, 1967), was subtracted from the total amount of cell-associated chloramphenicol, it appeared that chloramphenicol concentrations in the cell "pool" of E. coli and P. aeruginosa were respectively 136-3 and 96-5 times the concentration in the medium. Thus, these results clearly indicate that concentration of chloramphenicol by bacteria does not result simply from binding of the antibiotic to ribosomes, but that it is accumulated against a concentration gradient.…”

Section: Discussionmentioning

confidence: 99%

Transport of chloramphenicol into sensitive strains of Escherichia coli and Pseudomonas aeruginosa

Abdel-Sayed

1987

J Antimicrob Chemother

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The uptake of chloramphenicol by susceptible strains of Escherichia coli and Pseudomonas aeruginosa was measured as the depletion of 14 C-chloramphenicol from the supernatant of centrifuged cultures. Chloramphenicol did not bind to nongrowing cells or isolated cell envelopes. Chloramphenicol was recovered from cells in an unchanged form and was intracellularly concentrated several times above external concentrations. The net accumulation of the drug was reduced by an inhibitor of electron transport, by an oxidative phosphorylation uncoupler, by an inhibitor of high energy phosphate synthesis, and by lowering the temperature to + 15°C. The initial uptake of drug was saturated at 1 -98 mM chloramphenicol in the medium. A 100-fold excess of each of the unlabelled isomers: L-threo, D-threo, and L-erythro chloramphenicol in cultures of either strain effectively reduced the uptake of 14 C-chloramphenicol. These results indicate that chloramphenicol enters Gramnegative bacteria by means of an energy-dependent process.

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Mode of action of chloramphenicol IX. Effects of chloramphenicol upon a ribosomal amino acid polymerization system and its binding to bacterial ribosome

Cited by 131 publications

References 11 publications

Identification of Chloramphenicol-Binding Protein in Escherichia coli Ribosomes by Affinity Labeling

Identification of Chloramphenicol-Binding Protein in Escherichia coli Ribosomes by Affinity Labeling

Pleiotropic Antibiotic Resistance Mutations Associated with Ribosomes and Ribosomal Subunits in Mycobacterium smegmatis

Transport of chloramphenicol into sensitive strains of Escherichia coli and Pseudomonas aeruginosa

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