Kinetics and motility of the Eg5 microtubule motor
Kinetics and motility of the Eg5 microtubule motor
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DOI：
10.1021/bi952318n
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发表时间：
1996-02-20
期刊：
影响因子：
2.9
通讯作者：
Cross, RA
中科院分区：
文献类型：
--
作者：
Lockhart, A;Cross, RA
我们研究了慢加端定向微管（MT）电机Eg 5的动力学特性。重组表达的融合蛋白E437 GST含有融合到谷胱甘肽S-转移酶（GST）N-末端的Eg 5的残基12-437，是二聚体和能动的，以0.063（+/-0.01）μ m/s（-1）的平均速度易位MT。使用荧光ATP类似物甲基邻氨基苯甲酰-ATP（mantATP）研究E437 GST的ATP周转动力学。在没有MT的情况下，mantADP从E437 GST的释放是缓慢的（在50 mM NaCl中为0.006 s（-1））和限速的。MT使这一动力学步骤加速850倍，最大速率为4.94 s（-1）。在这些条件下，稳态mantATP周转率为1.92 s（-1），表明MT激活的mantADP释放至少占电机总循环时间的40%，可能是限速的。这一步在Eg 5中比驱动蛋白慢10倍左右，这与它限制Eg 5和驱动蛋白的物理步进速率一致。使用MT沉淀测定法测定了在各种核苷酸存在下马达的解离常数。ADP稳定马达的最弱结合状态，而ATP、ATP γ S、AMPPNP和腺苷三磷酸双磷酸酶都诱导向更紧密结合状态的转变。总体而言，数据表明，Eg 5显示出很强的动力学同源性与其他两个良好的特点MT电机，驱动蛋白和非红葡萄酒分离，这表明所有驱动蛋白超家族电机可能共享相同的基本机械化学。
We have investigated the kinetic properties of the slow plus end directed microtubule (MT) motor Eg5. The recombinantly expressed fusion protein E437GST, containing residues 12-437 of Eg5 fused to the N-terminus of glutathione S-transferase (GST), is dimeric and motile, translocating MTs at an average speed of 0.063 (+/-0.01) mu m s(-1). The kinetics of ATP turnover by E437GST were investigated using the fluorescent ATP analogue methylanthraniloyl-ATP (mantATP). In the absence of MTs, mantADP release from E437GST is slow (0.006 s(-1) in 50 mM NaCl) and rate-limiting. MTs accelerate this kinetic step similar to 850-fold to a maximal rate of 4.94 s(-1). Under these conditions, the steady-state rate of mantATP turnover was 1.92 s(-1), indicating that MT-activated mantADP release accounts for at least 40% of the total cycle time of the motor and is probably rate-limiting. This step is around 10-fold slower in Eg5 than in kinesin, consistent with it limiting the rate of physical stepping in both Eg5 and kinesin. The dissociation constants of the motor in the presence of various nucleotides were determined using MT pelleting assays. ADP stabilizes the weakest bound state of the motor, while ATP, ATP gamma S, AMPPNP, and apyrase all induce a shift toward tighter binding states. Overall, the data indicate that Eg5 displays strong kinetic homologies with the two other well-characterized MT motors, kinesin and non claret disjunctional, suggesting that all kinesin superfamily motors may share the same basic mechanochemistry.