MODIFICATION OF THE MICROTUBULE-BINDING AND ATPASE ACTIVITIES OF KINESIN BY N-ETHYLMALEIMIDE (NEM) SUGGESTS A ROLE FOR SULFHYDRYLS IN FAST AXONAL-TRANSPORT
MODIFICATION OF THE MICROTUBULE-BINDING AND ATPASE ACTIVITIES OF KINESIN BY N-ETHYLMALEIMIDE (NEM) SUGGESTS A ROLE FOR SULFHYDRYLS IN FAST AXONAL-TRANSPORT
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DOI：
10.1021/bi00449a008
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发表时间：
1989-11-14
期刊：
影响因子：
2.9
通讯作者：
BRADY, ST
中科院分区：
文献类型：
--
作者：
PFISTER, KK;WAGNER, MC;BRADY, ST
N-乙基马来酰亚胺是一种使蛋白质中的游离巯基烷基化的试剂，已被用于探测巯基在驱动蛋白中的作用，驱动蛋白是快速轴突运输中膜结合细胞器运动的马达蛋白。当鱿鱼轴浆灌注浓度高于0.5 mM的NEM，细胞器的运动在这两个顺行和逆行的方向停止，和囊泡保持连接到微管。将高度纯化的牛脑驱动蛋白与相似浓度的NEM孵育，改变了酶的微管刺激的ATP酶活性，并在ATP存在下促进驱动蛋白与微管的结合。这些结果表明，驱动蛋白上巯基的烷基化改变了蛋白质的构象，深刻地影响其与ATP和微管的相互作用。因此，NEM敏感的巯基，可以提供一个有价值的工具，为解剖的功能结构域的驱动蛋白分子，并了解这种酶的机械化学循环。
N-Ethylmaleimide, an agent which alkylates free sulfhydryls in proteins, has been used to probe the role of sulfhydryls in kinesin, a motor protein for the movement of membrane-bounded organelles in fast axonal transport. When squid axoplasm is perfused with concentrations of NEM higher than 0.5 mM, organelle movements in both the anterograde and retrograde directions cease, and the vesicles remain attached to microtubules. Incubation of highly purified bovine brain kinesin with similar concentrations of NEM modifies the enzyme''s microtubule-stimulated ATPase activity and promotes the binding of kinesin to microtubules in the presence of ATP. These results suggest that alkylation of sulfhydryls on kinesin alters the conformation of the protein in a manner that profoundly affects its interactions with ATP and microtubules. The NEM-sensitive sulfhydryls, therefore, may provide a valuable tool for the dissection of functional domains of the kinesin molecule and for understanding the mechanochemical cycle of this enzyme.