Stability of drug-induced tubulin rings by fluorescence correlation spectroscopy
Stability of drug-induced tubulin rings by fluorescence correlation spectroscopy
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DOI：
10.1021/bi026751q
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发表时间：
2003-02-11
期刊：
影响因子：
2.9
通讯作者：
Sackett, DL
中科院分区：
文献类型：
--
作者：
Boukari, H;Nossal, R;Sackett, DL
应用荧光相关光谱（FCS）来研究微管蛋白环的稳定性，这是由于三个长春花结构域结合肽-念珠藻素1，hemiasterlin，和多拉司他丁10的相互作用的α-微管蛋白二聚体。这些肽抑制微管蛋白聚合成微管，相反，诱导形成的单壁微管蛋白环的平均直径为23.8 nm的念珠藻素和44.6 nm的平均直径hemiasterlin和海兔毒素，如微摩尔药物微管蛋白样品的电子显微镜所示。然而，流体动力学直径和荧光颗粒的表观数量，从纳摩尔药物微管蛋白样品中获得的FCS测量值的分析确定，表明环的稳定性取决于药物和微管蛋白浓度的变化。即使微管蛋白浓度低至1 nM，念珠藻素-微管蛋白环似乎也是最稳定的，而半海星素-微管蛋白环是最不稳定的，即使在相对高的浓度（100 nM）下也解聚。相比之下，多拉司他汀-微管蛋白环表现出中等水平的稳定性，仅在微管蛋白浓度低于10 nM时显著解聚。我们还比较了稳定性的结果与细胞毒性测量采取的几个细胞系，并注意到细胞培养物中的药物的细胞毒性和相应的药物诱导的环的稳定性之间的粗略的相关性。
Fluorescence correlation spectroscopy (FCS) was applied to investigate the stability of tubulin rings that result from the interaction of alphabeta-tubulin dimers with three vinca domain-binding peptides-cryptophycin 1, hemiasterlin, and dolastatin 10. These peptides inhibit tubulin polymerization into microtubules and, instead, induce the formation of single-walled tubulin rings of 23.8 nm mean diameter for cryptophycin and 44.6 nm mean diameter for hemiasterlin and dolastatin, as revealed by electron microscopy on micromolar drug-tubulin samples. However, the hydrodynamic diameter and the apparent number of fluorescent particles, determined from analysis of FCS measurements obtained from nanomolar drug-tubulin samples, indicate variation in the stability of the rings depending on the drug and the tubulin concentration. Cryptophycin-tubulin rings appear to be the most stable even with tubulin concentration as low as 1 nM, whereas hemiasterlin-tubulin rings are the least, depolymerizing even at relatively high concentrations (100 nM). In contrast, the dolastatin-tubulin rings demonstrate an intermediate level of stability, depolymerizing significantly only at tubulin concentrations below 10 nM. We also compare the stability results with those of cytotoxicity measurements taken on several cell lines and note a rough correlation between the cytotoxicity of the drugs in cell cultures and the stability of the corresponding drug-induced rings.