Shimizu T, Marchese-Ragona S P, Johnson K A
Department of Molecular and Cell Biology, Pennsylvania State University, University Park 16802.
Biochemistry. 1989 Aug 22;28(17):7016-21. doi: 10.1021/bi00443a035.
The microtubule-dynein complex consisting of 22S dynein from Tetrahymena cilia and MAP-free microtubules was subjected to treatment with various concentrations of 1-ethyl-3-[3-(dimethylamino)-propyl]carbodiimide (EDC), a zero-length cross-linker, at 28 degrees C for 1 h. Following cross-linking of the microtubule-dynein complex, nearly all of the ATPase activity cosedimented with the microtubules in the presence of ATP. Electron microscopic observation by negative staining revealed that, following treatment with 1 mM EDC, the complex did not dissociate in the presence of ATP, although the dynein decoration pattern was disordered. The complex treated with 3 mM EDC exhibited normal microtubule-dynein patterns even after the addition of ATP. The ATPase activity of the microtubule-dynein complex was enhanced about 30-fold by the treatment with 1-3 mM EDC. These results indicate that the ATPase activation was caused by the close proximity of the dynein ATPase sites to the microtubules and provide further support for the functional interaction of all three dynein heads with the microtubule. The maximal specific activity was 12 mumol min-1 (mg of dynein)-1, corresponding to a turnover rate of 150 s-1, which may be the rate-limiting step at infinite microtubule concentration and may represent the maximum rate of force production in the axoneme.
由四膜虫纤毛的22S动力蛋白和无微管相关蛋白（MAP）的微管组成的微管 - 动力蛋白复合体，在28℃下用不同浓度的零长度交联剂1 - 乙基 - 3 - [3 - （二甲基氨基）丙基]碳二亚胺（EDC）处理1小时。微管 - 动力蛋白复合体交联后，在ATP存在的情况下，几乎所有的ATP酶活性都与微管一起沉降。负染电子显微镜观察显示，用1 mM EDC处理后，尽管动力蛋白的装饰模式紊乱，但复合体在ATP存在下并未解离。用3 mM EDC处理的复合体即使在添加ATP后仍表现出正常的微管 - 动力蛋白模式。用1 - 3 mM EDC处理使微管 - 动力蛋白复合体的ATP酶活性提高了约30倍。这些结果表明，ATP酶的激活是由于动力蛋白ATP酶位点与微管紧密接近所致，并为所有三个动力蛋白头部与微管的功能相互作用提供了进一步的支持。最大比活性为12 μmol min⁻¹（mg动力蛋白）⁻¹，对应于150 s⁻¹的周转速率，这可能是在无限微管浓度下的限速步骤，并且可能代表轴丝中力产生的最大速率。
