Beggs W H, Andrews F A
Antimicrob Agents Chemother. 1974 Mar;5(3):234-9. doi: 10.1128/AAC.5.3.234.
Recent studies showed that critical binding of ethambutol to Mycobacterium smegmatis was both inhibited and reversed by ions. This apparent ion-susceptible characteristic suggested that ethambutol is held at critical sites by electrostatic bonds. Dissociation constant, pH, and ionic strength studies were designed to further characterize ethambutol binding. M. smegmatis was grown in Sauton synthetic liquid medium (pH 7.4) under aerated conditions at 37 C. The pH or ionic strength of the medium was modified to meet the needs of particular experiments. Titration data revealed that ethambutol dihydrochloride has two apparent dissociation constants (pKa(1) = 6.35, pKa(2) = 9.35). Uptake experiments, in which pH was varied, showed that dihydrochloride and free base ethambutol were bound to a greater extent than the monohydrochloride. However, dihydrochloride and free base binding were not related to biological activity. Ethambutol exerted its maximal growth inhibitory effect at pH values near neutrality, where it exists primarily as the monohydrochloride and showed minimal binding. The increased ethambutol binding observed at pH 7.4 in media of lowered ionic strength was consistent with growth studies showing a reduction in the minimal inhibitory growth concentration in such media. However, nonspecific as well as critical binding was enhanced at low ionic strength. We conclude that binding of ethambutol by M. smegmatis involves a heterogeneous group of drug binding sites, only one of which is directly related to biological activity. Although nothing is known about the ethambutol target site itself, critical binding of the drug seems to require the single positively charged monohydrochloride form. Both hydrogen bonds and ionic linkages are probably involved.
最近的研究表明，离子会抑制并逆转乙胺丁醇与耻垢分枝杆菌的关键结合。这种明显的离子敏感性特征表明，乙胺丁醇通过静电键固定在关键位点上。设计了解离常数、pH值和离子强度研究，以进一步表征乙胺丁醇的结合情况。耻垢分枝杆菌在pH 7.4的索顿合成液体培养基中，于37℃通气条件下培养。根据特定实验的需要对培养基的pH值或离子强度进行调整。滴定数据显示，盐酸乙胺丁醇有两个明显的解离常数（pKa(1)=6.35，pKa(2)=9.35）。改变pH值的摄取实验表明，盐酸盐和游离碱形式的乙胺丁醇比一盐酸盐的结合程度更高。然而，盐酸盐和游离碱的结合与生物活性无关。乙胺丁醇在接近中性的pH值下发挥其最大的生长抑制作用，此时它主要以一盐酸盐形式存在，且结合最少。在离子强度降低的培养基中，pH 7.4时观察到的乙胺丁醇结合增加，与生长研究结果一致，即在这种培养基中最小抑菌生长浓度降低。然而，在低离子强度下，非特异性结合以及关键结合均增强。我们得出结论，耻垢分枝杆菌对乙胺丁醇的结合涉及一组异质的药物结合位点，其中只有一个与生物活性直接相关。尽管对乙胺丁醇的靶位点本身一无所知，但该药物的关键结合似乎需要带单个正电荷的一盐酸盐形式。氢键和离子键可能都参与其中。
