Interaction of p-benzoquinone and p-biphenoquinone with microtubule proteins in vitro
Interaction of p-benzoquinone and p-biphenoquinone with microtubule proteins in vitro
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DOI：
10.1016/0009-2797(96)03730-1
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发表时间：
1996-09-27
影响因子：
5.1
通讯作者：
Metzler, M
中科院分区：
文献类型：
--
作者：
Pfeiffer, E;Metzler, M
对苯二酚(BQ)和对联苯二酚(BPQ)是人骨髓毒素和白血病原苯的代谢产物，已被报道在哺乳动物细胞中诱导非整倍体。由于苯的非整倍体效应的一个可能机制可能是通过BQ和BPQ与微管蛋白(MTP)的共价结合而破坏有丝分裂纺锤体，我们研究了在无细胞条件下这些苯醌与MTP的反应及其对微管形成的影响。BQ和BPQ均以浓度依赖的方式抑制MTP与MT的组装。这种相互作用伴随着对苯二酚的光谱变化和MTP的自由巯基的损失。用40mU的M-BQ或BPQ，MT的组装被观察到50%的抑制，并伴随着每个微管蛋白二聚体失去1.3个硫醇基团。进一步的分析表明，天然MTP与BQ和BPQ都形成了单加合物，但没有二加成，也没有二硫键。通过对碱性过甲基化生成的不同硫代苯甲醇的GC/MS分析，明确地证明了共价醌/MTP单加合物的形成。变性的MTP、谷胱甘肽或2-巯基乙醇与BQ形成几乎排他的单加合物，但与BPQ形成高比例的二硫键。因此，BQ和BPQ与能够形成二硫键的硫醇化合物反应不同。这两种醌只与天然MTP形成单加合物的事实可以用天然微管蛋白的硫醇基团不容易形成氧化二硫键的假设来解释。在导致谷胱甘肽完全氧化的条件下，用过氧化氢/辣根过氧化物酶处理缺乏形成二硫键的天然MTP，这支持了这一命题。苯代谢物BQ和BPQ与微管蛋白的关键硫醇基团的共价结合抑制了无细胞条件下MT的形成，并可能干扰有丝分裂细胞中功能纺锤体的形成，从而导致已报道的苯的异常染色体分离和非整倍体诱导。
p-benzoquinone (BQ) and p-biphenoquinone (BPQ) are metabolites of the human myelotoxin and leukemogen benzene, which has been reported to induce aneuploidy in mammalian cells. Because a possible mechanism for the aneuploidogenic effect of benzene may be the disruption of the mitotic spindle by covalent binding of BQ and BPQ to microtubule proteins (MTP), we have studied the reaction of these quinones with MTP and its consequences for microtubule (MT) formation under cell-free conditions. Both BQ and BPQ inhibit the assembly of MTP to MT in a concentration-dependent manner. This interaction is accompanied by a spectral change of the quinones and loss of free sulfhydryl groups of MTP. With 40 mu M BQ or BPQ, 50% inhibition of MT assembly was observed and associated with the loss of 1.3 thiol groups per tubulin dimer. Further analysis showed that native MTP form monoadducts, but no diadducts nor disulfide bonds with both BQ and BPQ. The formation of covalent quinone/MTP monoadducts was unequivocally demonstrated by GC/MS analysis of the respective thioanisols liberated by alkaline permethylation. Denatured MTP or glutathione or 2-mercaptoethanol gave rise to the virtually exclusive formation of monoadducts with BQ but led to a high proportion of disulfide bonds with BPQ. Therefore, BQ and BPQ react differently with thiol compounds capable of disulfide bond formation. The fact that both quinones form only monoadducts with native MTP can be explained by the assumption that the thiol groups of native tubulin are not prone to oxidative disulfide bond formation. This proposition was supported by the lack of native MTP to form disulfide bridges upon treatment with hydrogen peroxide/horseradish peroxidase under conditions leading to a complete oxidation of glutathione. The covalent binding of the benzene metabolites BQ and BPQ to critical thiol groups of tubulin inhibits MT formation under cell-free conditions and may also interfere with the formation of a functional spindle apparatus in the mitotic cell, thus leading to the abnormal chromosome segregation and aneuploidy induction reported for benzene.