Moss A G, Gatti J L, Witman G B
Cell Biology Group and Male Fertility Program, Worcester Foundation for Experimental Biology, Shrewsbury, Massachusetts 01545.
J Cell Biol. 1992 Sep;118(5):1177-88. doi: 10.1083/jcb.118.5.1177.
We used in vitro translocation and cosedimentation assays to study the microtubule binding properties of sea urchin sperm outer arm dynein and its beta/IC1 subunit. Microtubules glided on glass-absorbed sea urchin dynein for a period of time directly proportional to the initial MgATP2- concentration and then detached when 70-95% of the MgATP2- was hydrolyzed. Detachment resulted from MgATP2- depletion, because (a) perfusion with fresh buffer containing MgATP2- reconstituted binding and gliding, (b) microtubules glided many minutes with an ATP-regenerating system at ATP concentrations which alone supported gliding for only 1-2 min, and (c) microtubules detached upon total hydrolysis of ATP by an ATP-removal system. The products of ATP hydrolysis antagonized binding and gliding; as little as a threefold excess of ADP/Pi over ATP resulted in complete loss of microtubule binding and translocation by the beta/IC1 subunit. In contrast to the situation with sea urchin dynein, microtubules ceased gliding but remained bound to glass-absorbed Tetrahymena outer arm dynein when MgATP2- was exhausted. Cosedimentation assays showed that Tetrahymena outer arm dynein sedimented with microtubules in an ATP-sensitive manner, as previously reported (Porter, M.E., and K. A. Johnson. J. Biol. Chem. 258: 6575-6581). However, the beta/IC1 subunit of sea urchin dynein did not cosediment with microtubules in the absence of ATP. Thus, this subunit, while capable of generating motility, lacks both structural and rigor-type microtubule binding.
我们使用体外转运和共沉降分析来研究海胆精子外臂动力蛋白及其β/IC1亚基的微管结合特性。微管在玻璃吸附的海胆动力蛋白上滑动一段时间，该时间与初始MgATP2-浓度成正比，然后在70-95%的MgATP2-被水解时脱离。脱离是由于MgATP2-耗尽，因为：(a) 用含有MgATP2-的新鲜缓冲液灌注可恢复结合和滑动；(b) 微管在ATP再生系统存在下，在仅能支持1-2分钟滑动的ATP浓度下滑动了许多分钟；(c) 微管在ATP去除系统将ATP完全水解后脱离。ATP水解产物拮抗结合和滑动；ADP/Pi比ATP仅过量三倍就导致β/IC1亚基完全丧失微管结合和转运能力。与海胆动力蛋白的情况相反，当MgATP2-耗尽时，微管停止滑动但仍与玻璃吸附的四膜虫外臂动力蛋白结合。共沉降分析表明，四膜虫外臂动力蛋白以ATP敏感的方式与微管一起沉降，如先前报道的那样（波特，M.E.，和K.A.约翰逊。《生物化学杂志》258: 6575-6581）。然而，在没有ATP的情况下，海胆动力蛋白的β/IC1亚基不会与微管共沉降。因此，该亚基虽然能够产生运动，但缺乏结构和强直型微管结合能力。
