Chang F N, Flaks J G
Antimicrob Agents Chemother. 1972 Oct;2(4):294-307. doi: 10.1128/AAC.2.4.294.
The binding of dihydrostreptomycin to ribosomes and ribosomal subunits of a number of different Escherichia coli strains was studied, and the Mg(2+) and pH dependence, as well as the effect of salts and polynucleotides, was determined. The only requirement for binding with ribosomes and subunits from susceptible strains was 10 mm Mg(2+). Monovalent salts weakened the binding in a manner similar to the effects on ribonucleic acid secondary structure, and this was antagonized to some extent by increased amounts of Mg(2+). Bound dihydrostreptomycin could be readily exchanged by streptomycin and any antibiotically active derivative, but not by fragments of the antibiotic or any other aminoglycoside. With native (run-off) 70S ribosomes from streptomycin-susceptible strains, the binding was rapid and relatively temperature independent over the range from 0 to 37 C. Polynucleotides did not stimulate the binding. With concentrations of dihydrostreptomycin up to 10(-5)m, greater than 95% of native 70S ribosomes bound exactly 1 molecule of the antibiotic tightly, with a K(diss) for the bound complex at 25 C of 9.4 x 10(-8)m. The following thermodynamic parameters were found for the binding with 70S ribosomes at 25 C:DeltaG degrees = -9.6 kcal/mole, DeltaH degrees = -6.2 kcal/mole, and DeltaS degrees = +11.4 entropy units/mole. Differences in affinity for the antibiotic were found between ribosomes of K-12 strains and those of other E. coli strains. There was insignificant binding to 70S ribosomes or subunits from streptomycin-resistant or -dependent strains, and to 50S subunits from susceptible strains. The binding to 30S subunits from susceptible strains was weaker by an order of magnitude than that to the 70S particle, with a K(diss) at 25 C of 10(-6)m. Polyuridylic acid stimulated this binding slightly but did not influence the affinity of the bound molecule. At antibiotic concentrations above 10(-5)m, streptomycin-susceptible 70S and 30S particles bound additional molecules of the antibiotic, and binding also occurred to ribosomes from streptomycin-resistant and -dependent strains, as well as to 50S subunits from all strains. K(diss) for all of these binding equilibria were [Formula: see text] 10(-4)m. This weaker non-specific binding coincided with the beginning of aggregation phenomena involving the particles, and occurred at sites distinct from the single site which binds the antibiotic tightly. This latter site was completely lost after the one-step mutation to high-level resistance or dependence.
研究了双氢链霉素与多种不同大肠杆菌菌株的核糖体及核糖体亚基的结合情况，并测定了镁离子（Mg²⁺）浓度、pH值依赖性以及盐类和多核苷酸的影响。与敏感菌株的核糖体及亚基结合的唯一条件是存在10 mM的Mg²⁺。单价盐以类似于对核糖核酸二级结构影响的方式减弱结合，而增加Mg²⁺的量在一定程度上可对抗这种作用。结合的双氢链霉素可被链霉素及任何具有抗菌活性的衍生物轻易置换，但不能被抗生素片段或任何其他氨基糖苷类置换。对于来自链霉素敏感菌株的天然（连续转录）70S核糖体，在0至37℃范围内结合迅速且相对不依赖温度。多核苷酸不会刺激结合。当双氢链霉素浓度高达10⁻⁵ M时，超过95%的天然70S核糖体紧密结合恰好1分子抗生素，在25℃时结合复合物的解离常数（K(diss)）为9.4×10⁻⁸ M。在25℃下与70S核糖体结合时发现以下热力学参数：ΔG° = -9.6 kcal/摩尔，ΔH° = -6.2 kcal/摩尔，ΔS° = +11.4熵单位/摩尔。发现K-12菌株的核糖体与其他大肠杆菌菌株的核糖体对该抗生素的亲和力存在差异。与链霉素抗性或依赖性菌株的70S核糖体或亚基以及敏感菌株的50S亚基几乎没有结合。与敏感菌株的30S亚基的结合比与70S颗粒的结合弱一个数量级，在25℃时的K(diss)为10⁻⁶ M。聚尿苷酸略微刺激这种结合，但不影响结合分子的亲和力。在抗生素浓度高于10⁻⁵ M时，链霉素敏感的70S和30S颗粒结合额外的抗生素分子，并且链霉素抗性和依赖性菌株的核糖体以及所有菌株的50S亚基也会发生结合。所有这些结合平衡的K(diss)均≥10⁻⁴ M。这种较弱的非特异性结合与涉及颗粒的聚集现象开始同时出现，并且发生在与紧密结合抗生素的单个位点不同的位点。在一步突变至高水平抗性或依赖性后，后一个位点完全丧失。
