Fok A K, Wang H, Katayama A, Aihara M S, Allen R D
Pacific Biomedical Research Center, University of Hawaii Honolulu 96822.
Cell Motil Cytoskeleton. 1994;29(3):215-24. doi: 10.1002/cm.970290304.
In an earlier study we reported the isolation of a cytoplasmic dynein from the cytosol of Paramecium multimicronucleatum. In this study we report the isolation and characterization of two cytosolic axonemal dyneins (22S and 12S) as well as a 19S cytoplasmic dynein from the cytosol of whole or deciliated cells using preformed bovine brain microtubules. These three dynein species were characterized according to mass, morphology, vanadate photocleavage patterns, CTPase/ATPase ratios, Km and Vmax values, temperature optima and reactivity with a mAb. For comparison, 22S and 12S axonemal dyneins (ADs) were also isolated and purified from the demembranated axonemes. The 22S and 12S soluble dyneins appear to be related to ciliary ADs in that the 22S soluble dynein is three-headed while the 12S is a one-headed dynein, as determined by negative staining. Ciliary ADs and their corresponding 22S and 12S soluble dyneins isolated from the cytosol also have similar Km and Vmax values as well as vanadate photocleavage patterns and temperature optima. A mAb raised against the soluble 22S dynein reacted with the 22S ciliary dyneins but not the 12S axonemal or the 19S cytoplasmic dynein. All isolated dyneins supported similar microtubule gliding rates but had different ionic requirements for the translocation buffer. These results suggest that: (i) the two soluble 22S and 12S dyneins are precursor molecules of the ciliary dyneins, (ii) the subunits of the outer arm dynein are already assembled in the cytosol as a three-headed bouquet, and (iii) the 22S and 12S soluble dyneins are functional prior to being transported and attached to the axonemes of the cilia.
在早期的一项研究中，我们报道了从多核草履虫的胞质溶胶中分离出一种胞质动力蛋白。在本研究中，我们报道了使用预先形成的牛脑微管，从完整或去纤毛细胞的胞质溶胶中分离并鉴定了两种胞质轴丝动力蛋白（22S和12S）以及一种19S胞质动力蛋白。根据质量、形态、钒酸盐光裂解模式、CTP酶/ATP酶比率、Km和Vmax值、最适温度以及与单克隆抗体的反应性对这三种动力蛋白进行了表征。为了进行比较，还从去膜轴丝中分离并纯化了22S和12S轴丝动力蛋白（ADs）。通过负染色确定，22S和12S可溶性动力蛋白似乎与纤毛ADs相关，因为22S可溶性动力蛋白是三头的，而1..S是单头动力蛋白。从胞质溶胶中分离出的纤毛ADs及其相应的22S和12S可溶性动力蛋白也具有相似的Km和Vmax值，以及钒酸盐光裂解模式和最适温度。针对可溶性22S动力蛋白产生的单克隆抗体与22S纤毛动力蛋白反应，但不与12S轴丝动力蛋白或19S胞质动力蛋白反应。所有分离出的动力蛋白都支持相似的微管滑动速率，但对转运缓冲液有不同的离子需求。这些结果表明：（i）两种可溶性22S和12S动力蛋白是纤毛动力蛋白的前体分子；（ii）外臂动力蛋白的亚基在胞质溶胶中已经组装成三头花束状；（iii）22S和12S可溶性动力蛋白在被运输并附着到纤毛的轴丝之前就已经具有功能。
