IDENTIFICATION OF CHLORAMPHENICOL-BINDING PROTEIN IN ESCHERICHIA-COLI RIBOSOMES BY AFFINITY LABELING
IDENTIFICATION OF CHLORAMPHENICOL-BINDING PROTEIN IN ESCHERICHIA-COLI RIBOSOMES BY AFFINITY LABELING
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DOI：
10.1073/pnas.70.8.2229
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发表时间：
1973-01-01
影响因子：
11.1
通讯作者：
ERDMANN, VA
中科院分区：
文献类型：
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作者：
PONGS, O;BALD, R;ERDMANN, VA
氯霉素和林可霉素的竞争实验表明，氯霉素的合成类似物单碘氯霉素与氯霉素结合在70 S核糖体的相同位点。在-2 °时，它与70 S核糖体形成1：1的复合物，其K值为7.5 × 104 M-1，比氯霉素低一个数量级。在37 ℃时，单碘氯霉素不可逆地抑制E.大肠核糖体结果表明，该类似物优先与大肠杆菌L16蛋白反应。coli 70 S核糖体的结合位点，因此我们认为L16蛋白属于氯霉素结合位点。由于单碘氯霉素的化学反应基团类似于碘乙酰胺，因此E. coli 70 S核糖体与单碘氯霉素和碘乙酰胺的比较。碘乙酰胺不与蛋白质L16反应，但它主要与30 S亚基的蛋白质S18反应。此外，单碘氯霉素与E. coliribosomal亚单位分离的50 S亚基结合monoiodoamphenicol约一个数量级小于70 S核糖体。再次，蛋白L16与亲和标记反应。Monoiodoamphenicol与分离的30 S亚基中的蛋白S18反应，它也与嗜热脂肪芽孢杆菌的70 S核糖体结合，然而，它不与这些70 S核糖体不可逆地结合。
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 ofK(7.5 × 104M-1) that is one order of magnitude lower than that of chloramphenicol. At 37°, monoiodoamphenicol irreversibly inhibits the protein-synthesizing activity ofE. coliribosomes. It is shown that the analogue reacted preferentially with protein L16 ofE. coli70S ribosomes, and we therefore conclude that protein L16 belongs to the chloramphenicol-binding site ofE. coliribosomes.Since the chemically reactive group of monoiodoamphenicol resembles iodoacetamide, the reaction ofE. coli70S 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 withE. coliribosomal 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 ofBacillus stearothermophilus, however, it did not bind irreversibly to these 70S ribosomes.