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首页> 外文期刊>Biochemistry >Pathway of ADP-stimulated ADP release and dissociation of tethered kinesin from microtubules. Implications for the extent of processivity.
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Pathway of ADP-stimulated ADP release and dissociation of tethered kinesin from microtubules. Implications for the extent of processivity.

机译：ADP刺激的ADP释放和束缚驱动蛋白从微管解离的途径。对生产能力的影响。

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摘要

Kinesin binds to microtubules with half-site ADP release to form a tethered intermediate with one attached head without nucleotide and one tethered head that retains its bound ADP. For DKH405 containing amino acid residues 1-405 of Drosophila kinesin, release of the remaining ADP from the tethered head is slow (0.05 s(-1)), but release is accelerated by added ADP or ATP. The maximum rate of ADP-stimulated dissociation of tethered DKH405 from the microtubule is approximately 12 s(-1) as determined by turbidity. Parallel measurements of ADP-stimulated release of 2'(3')-O-(N-methylanthraniloyl)-ADP (mantADP) from the tethered intermediate by fluorescence indicate that the reaction is biphasic with a fast phase that occurs at a rate that is similar to dissociation. The rate of the slow phase is dependent on the concentrations of salt and microtubules and is equal in each case to the rate for bimolecular stimulation of ADP release by microtubules as measured independently. These results are consistent with a scheme in which the fast phase, with approximately one-third of the total amplitude change, is due to ADP-stimulated release of mantADP from the tethered intermediate at approximately 6 s(-1). This direct release of mantADP continues until terminated by dissociation of DKH405 from the microtubule at approximately 12 s(-1). The majority of the amplitude change thus occurs through bimolecular recombination of DKH405.mantADP with microtubules following initial dissociation. Analysis of a simple scheme indicates that hydrolysis of ATP at the attached head before the tethered head can release its ADP and become tightly bound may be the principal limitation to processivity.
机译：驱动蛋白结合具有半位ADP释放的微管以形成一种束缚的中间体，该束缚的中间体具有一个没有核苷酸的附着头和一个保留其结合的ADP的束缚头。对于含有果蝇驱动蛋白氨基酸残基1-405的DKH405，从束缚的头部释放剩余的ADP的速度很慢（0.05 s（-1）），但是通过添加ADP或ATP可以加速释放。根据浊度确定，ADP刺激的DKH405从微管的解离的最大速率约为12 s（-1）。 ADP刺激通过荧光从束缚的中间体对ADP刺激的2'（3'）-O-（N-甲基蒽基）-ADP（mantADP）释放的平行测量表明该反应是双相的快速相，其发生速率为类似于解离。慢相的速率取决于盐和微管的浓度，并且在每种情况下均等于通过微管独立测量的双分子刺激ADP释放的速率。这些结果与一种方案相吻合，在该方案中，大约占总振幅变化三分之一的快速相位是由于ADP刺激了大约6 s（-1）从束缚的中间体释放了mantADP。 mantADP的直接释放一直持续到DKH405在大约12 s（-1）时从微管解离终止。因此，在初始解离后，大多数振幅变化是通过DKH405.mantADP与微管的双分子重组发生的。对简单方案的分析表明，在束缚的头能够释放其ADP并紧密结合之前，连接头上的ATP水解可能是合成能力的主要限制。

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