Foster K A, Correia J J, Gilbert S P
Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania 15260, USA.
J Biol Chem. 1998 Dec 25;273(52):35307-18. doi: 10.1074/jbc.273.52.35307.
Non-claret disjunctional protein (Ncd) is a minus end-directed microtubule motor required for normal spindle assembly and integrity during Drosophila oogenesis. We have pursued equilibrium binding experiments to examine the affinity of Ncd for microtubules in the presence of the ATP nonhydrolyzable analog 5'-adenylyl-beta, gamma-imidodiphosphate (AMP-PNP), ADP, or ADP + Pi using both dimeric (MC1) and monomeric (MC6) Ncd constructs expressed in Escherichia coli. Both MC1 and MC6 sediment with microtubules in the absence of added nucleotide as well as in the presence of either ADP or AMP-PNP. Yet, in the presence of ADP + Pi, there is a decrease in the affinity of both MC1 and MC6 for microtubules. The data for dimeric MC1 show that release of the dimer to the supernatant is sigmoidal with the apparent Kd(Pi) for the two phosphate sites at 23.3 and 1.9 mM, respectively. The results indicate that binding at the first phosphate site enhances binding at the second site, thus cooperatively stimulating release. Stopped-flow kinetics indicate that MgATP promotes dissociation of the Mt.MC1 complex at 14 s-1, yet AMP-PNP has no effect on the Mt.MC1 complex. These results are consistent with a model for the ATPase cycle in which ATP hydrolysis occurs on the microtubule followed by detachment as the Ncd.ADP.Pi intermediate.
非紫红色分离蛋白（Ncd）是一种负端定向微管马达蛋白，在果蝇卵子发生过程中对正常纺锤体组装和完整性至关重要。我们进行了平衡结合实验，以研究在ATP不可水解类似物5'-腺苷-β，γ-亚氨基二磷酸（AMP-PNP）、ADP或ADP + Pi存在的情况下，使用在大肠杆菌中表达的二聚体（MC1）和单体（MC6）Ncd构建体时，Ncd与微管的亲和力。在没有添加核苷酸以及存在ADP或AMP-PNP的情况下，MC1和MC6均与微管一起沉降。然而，在存在ADP + Pi的情况下，MC1和MC6与微管的亲和力均降低。二聚体MC1的数据表明，二聚体释放到上清液呈S形，两个磷酸位点的表观Kd（Pi）分别为23.3和1.9 mM。结果表明，在第一个磷酸位点的结合增强了在第二个位点的结合，从而协同刺激释放。停流动力学表明，MgATP以14 s-1的速度促进Mt.MC1复合物的解离，但AMP-PNP对Mt.MC1复合物没有影响。这些结果与ATPase循环模型一致，在该模型中，ATP水解发生在微管上，随后作为Ncd.ADP.Pi中间体脱离。
