Martenson C H, Odom A, Sheetz M P, Graham D G
Department of Pathology, Duke University Medical Center, Durham, North Carolina 27710, USA.
Toxicol Appl Pharmacol. 1995 Jul;133(1):73-81. doi: 10.1006/taap.1995.1128.
Chronic exposure to acrylamide leads to a dying-back axonopathy afflicting the longest axons of all tested mammalian and avian species. Prior to the onset of acrylamide-induced axonal degeneration, alterations in axonal fast transport have been consistently reported to be more severe for the retrograde than the anterograde direction. The putative retrograde motor protein, dynein, is compromised by exposure to the sulfhydryl-alkylating agent N-ethylmaleimide (NEM) at concentrations far below those required to inactivate kinesin, the putative anterograde motor protein. Since acrylamide is capable of alkylating protein sulfhydryl moieties, we tested whether a direct exposure of purified kinesin or dynein to acrylamide would result in an impairment of either enzyme's ability to translocate microtubules. Motor activity was assayed by sequentially adsorbing either kinesin or dynein to acid-washed coverslips, treating with an alkylating agent or control solution, adding microtubules and ATP, and finally imaging and quantifying the binding and gliding of microtubules using video-enhanced differential interference contrast (VE-DIC) microscopy. In comparison to controls, incubation of dynein with NEM, ethacrynic acid, or iodoacetic acid resulted in dose-dependent decreases in the amount and rate of microtubule gliding, but increases in irreversible high-affinity microtubule binding. In contrast, exposure of dynein to 1-100 mM solutions of acrylamide did not significantly alter either the binding or gliding of microtubules (a molar/hour exposure to acrylamide equivalent to 50 times that which causes retrograde transport deficits in vivo). Likewise, kinesin motility parameters were not significantly affected by acrylamide concentrations up to 100 mM while NEM solutions > 100 microM led to significant losses in the ability of kinesin to bind MT. These data indicate that acrylamide does not significantly interact with bound (adsorbed) kinesin or dynein, implying that the mechanism by which acrylamide interferes with fast axonal transport in vivo is by interaction with other factor(s) that govern the movement of vesicles.
长期接触丙烯酰胺会导致一种轴突逆行性病变，影响所有受试哺乳动物和鸟类物种中最长的轴突。在丙烯酰胺诱导的轴突变性开始之前，一直有报道称轴突快速运输的改变在逆行方向比顺行方向更严重。假定的逆行运动蛋白动力蛋白，在远低于使假定的顺行运动蛋白驱动蛋白失活所需浓度的情况下，会因暴露于巯基烷基化剂N - 乙基马来酰亚胺（NEM）而受损。由于丙烯酰胺能够使蛋白质巯基部分烷基化，我们测试了将纯化的驱动蛋白或动力蛋白直接暴露于丙烯酰胺是否会导致这两种酶转运微管的能力受损。通过依次将驱动蛋白或动力蛋白吸附到酸洗盖玻片上，用烷基化剂或对照溶液处理，添加微管和ATP，最后使用视频增强微分干涉对比（VE - DIC）显微镜对微管的结合和滑动进行成像和定量来测定运动活性。与对照相比，动力蛋白与NEM、依他尼酸或碘乙酸孵育会导致微管滑动的量和速率呈剂量依赖性降低，但不可逆的高亲和力微管结合增加。相比之下，将动力蛋白暴露于1 - 100 mM的丙烯酰胺溶液中，并未显著改变微管的结合或滑动（每小时丙烯酰胺暴露量相当于体内导致逆行运输缺陷量的50倍）。同样，高达100 mM的丙烯酰胺浓度对驱动蛋白的运动参数没有显著影响，而>100 microM的NEM溶液会导致驱动蛋白结合微管的能力显著丧失。这些数据表明丙烯酰胺不会与结合（吸附）的驱动蛋白或动力蛋白发生显著相互作用，这意味着丙烯酰胺在体内干扰快速轴突运输的机制是与控制囊泡运动的其他因素相互作用。
