KINESIN ATPASE - RATE-LIMITING ADP RELEASE
KINESIN ATPASE - RATE-LIMITING ADP RELEASE
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DOI：
10.1073/pnas.85.17.6314
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发表时间：
1988-09-01
影响因子：
11.1
通讯作者：
HACKNEY, DD
中科院分区：
文献类型：
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作者：
HACKNEY, DD
用S. A. Kuznetsov和V.I. Gelfand [（1986）Proc.Natl. Acad. Sci. USA 83，8530-8534）]通过与微管蛋白的相互作用而被刺激1000倍（每120-kDa肽的周转率从约1000- 1000倍增加到约1000倍）。0.009秒-1至9秒-1）。微管蛋白刺激的反应表现出没有额外的掺入水衍生的氧在很宽的范围内的ATP和微管蛋白的浓度，表明Pi的释放比水解的逆转更快。然而，ADP释放对于基础反应是缓慢的，并且其释放是速率限制性的，如通过非常紧密的ADP结合（Ki < 5 nM）、通过离子交换色谱和透析的化学计量水平的结合ADP的保留、以及如通过离心凝胶过滤和丙酮酸激酶的不可接近性所示的在稳态ATP酶速率下通过[14 C]ATP的结合ADP的可逆标记所指示的。微管蛋白加速结合ADP的释放，这与其激活净ATP酶反应一致。在微管蛋白存在下ADP释放的详细动力学是双相的，表明具有明显的异质性，其中一部分驱动蛋白活性位点不受微管蛋白的影响。
The ATPase rate of kinesin isolated from bovine brain by the method of S. A. Kuznetsov and V. I. Gelfand [(1986) Proc. Natl. Acad. Sci. USA 83, 8530-8534)] is stimulated 1000-fold by interaction with tubulin (turnover rate per 120-kDa peptide increases from .apprxeq. 0.009 sec-1 to 9 sec-1). The tubulin-stimulated reaction exhibits no extra incorporation of water-derived oxygens over a wide range of ATP and tubulin concentrations, indicating that Pi release is faster than the reversal of hydrolysis. ADP release, however, is slow for the basal reaction and its release is rate limiting as indicated by the very tight ADP binding (Ki < 5 nM), the retention of a stoichiometric level of bound ADP through ion-exchange chromatography and dialysis, and the reversible labeling of a bound ADP by [14C]ATP at the steady-state ATPase rate as shown by centrifuge gel filtration and inaccessibility to pyruvate kinase. Tubulin accelerates the release of the bound ADP consistent with its activation of the net ATPase reaction. The detailed kinetics of ADP release in the presence of tubulin are biphasic indicating apparent heterogeneity with a fraction of the kinesin active sites being unaffected by tubulin.