Identification of chloramphenicol-binding protein in Escherichia coli ribosomes by affinity labeling.
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 degrees it forms a 1:1 complex with 70S ribosomes having a value of K (7.5 x 10(4) M(-1)) that is one order of magnitude lower than that of chloramphenicol. At 37 degrees , 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 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.
D000081:Acetamides / Q000378:metabolism; D001345:Autoradiography; D001407:Bacillus / Q000378:metabolism; D001426:Bacterial Proteins / Q000302:isolation & purification* / Q000378:metabolism; D001665:Binding Sites; D001667:Binding, Competitive; D001966:Bromine / Q000378:metabolism; D002247:Carbon Isotopes; D002701:Chloramphenicol / Q000378:metabolism* / Q000494:pharmacology / Q000031:analogs & derivatives; D004926:Escherichia coli / Q000032:analysis* / Q000378:metabolism; D007455:Iodine / Q000378:metabolism; D007700:Kinetics; D010649:Phenylalanine / Q000096:biosynthesis; D011119:Polynucleotides / Q000378:metabolism; D011485:Protein Binding; D012270:Ribosomes / Q000032:analysis / Q000378:metabolism*
D000081:Acetamides; D001426:Bacterial Proteins; D002247:Carbon Isotopes; D011119:Polynucleotides; C005750:monoiodoamphenicol; D010649:Phenylalanine; D002701:Chloramphenicol; D007455:Iodine; D001966:Bromine