Shimizu T, Johnson K A
J Biol Chem. 1983 Nov 25;258(22):13841-6.
We have examined the kinetics of ATP-induced dissociation of the microtubule-dynein complex at low ATP concentrations in the presence of vanadate, which inhibits the enzyme after the binding and hydrolysis of a single ATP per site (Shimizu, T., and Johnson, K. A. (1983) J. Biol. Chem. 258, 13833-13840). Four aspects of the dissociation reaction could not be explained by a model of dynein with a single ATP-sensitive microtubule binding site. First, titration of the light-scattering amplitude versus ATP concentration in the presence of vanadate gave Mr = 720,000/ATP binding site, indicating approximately 2.8 sites/2 million molecular weight particle. Second, the dissociation reaction was incomplete at concentrations of less than 2 microM ATP in the absence of vanadate, while the addition of vanadate led to complete dissociation at an increased rate. Third, the time course of dissociation induced by less than or equal to 1 microM ATP in the presence of vanadate was biphasic, with a small but distinct lag. Fourth, the ATP concentration dependence of the rate of dissociation in the absence of vanadate was concave upward at concentrations of ATP less than 5 microM, whereas the plot was linear in the presence of vanadate. These data suggest that dynein has three ATP-sensitive microtubule binding sites and each site must bind ATP for dynein to detach from the microtubule.
我们研究了在钒酸盐存在下低ATP浓度时ATP诱导的微管-动力蛋白复合体解离的动力学，钒酸盐在每个位点结合并水解一个ATP后抑制该酶（清水，T.，和约翰逊，K. A.（1983年）《生物化学杂志》258，13833 - 13840）。解离反应的四个方面无法用具有单个ATP敏感微管结合位点的动力蛋白模型来解释。首先，在钒酸盐存在下，光散射幅度对ATP浓度的滴定得出Mr = 720,000/ATP结合位点，表明每200万分子量的颗粒约有2.8个位点。其次，在没有钒酸盐的情况下，ATP浓度低于2 microM时解离反应不完全，而加入钒酸盐会导致以更快的速度完全解离。第三，在钒酸盐存在下，由小于或等于1 microM ATP诱导的解离时间进程是双相的，有一个小但明显的延迟。第四，在没有钒酸盐的情况下，ATP浓度低于5 microM时解离速率对ATP浓度的依赖性呈向上凹形，而在钒酸盐存在下该图是线性的。这些数据表明动力蛋白有三个ATP敏感的微管结合位点，并且每个位点必须结合ATP动力蛋白才能从微管上脱离。
