Hackney D D
Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA 15213.
Proc Natl Acad Sci U S A. 1988 Sep;85(17):6314-8. doi: 10.1073/pnas.85.17.6314.
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 approximately equal to 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 less than 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.
通过S.A. 库兹涅佐夫和V.I. 盖尔方德的方法（[(1986) 《美国国家科学院院刊》83, 8530 - 8534]）从牛脑中分离出的驱动蛋白的ATP酶速率，通过与微管蛋白相互作用可被刺激1000倍（每120 kDa肽的周转速率从约0.009秒⁻¹增加到9秒⁻¹）。在广泛的ATP和微管蛋白浓度范围内，微管蛋白刺激的反应未显示出额外掺入来自水的氧，这表明磷酸根离子（Pi）的释放比水解的逆转更快。然而，对于基础反应，ADP的释放很慢，并且其释放是限速步骤，这由非常紧密的ADP结合（解离常数Ki小于5 nM）、通过离子交换色谱和透析保留化学计量水平的结合ADP以及在稳态ATP酶速率下[¹⁴C]ATP对结合ADP的可逆标记所表明，如离心凝胶过滤所示且丙酮酸激酶无法作用于该结合ADP。微管蛋白加速结合ADP的释放，这与其对净ATP酶反应的激活一致。在微管蛋白存在下ADP释放的详细动力学是双相的，表明存在明显的异质性，一部分驱动蛋白活性位点不受微管蛋白影响。
