Faire K, Bonder E M
Department of Biological Sciences, Rutgers, State University of New Jersey, Newark 07102.
Dev Biol. 1993 Oct;159(2):581-94. doi: 10.1006/dbio.1993.1266.
We have initiated a study to identify possible tubulin dimer binding proteins from unfertilized sea urchin eggs utilizing tubulin dimer coupled to Sepharose 4B. A 100-kDa polypeptide has been isolated and its interaction with tubulin/microtubules examined. Solution assembly studies indicated the 100-kDa polypeptide potentiated microtubule assembly. The 100-kDa protein also bundled and cosedimented with polymerized microtubules. Rabbit polyclonal anti-egg 100-kDa antibodies cross-reacted with a 100-kDa polypeptide in the eggs of several different sea urchin species and with mammalian brain dynamin, a 100-kDa microtubule binding protein. These biochemical characterizations and immunological results suggest that the sea urchin 100-kDa microtubule binding protein appears to be related to the dynamin protein family. To gain insight into the functional role of sea urchin egg 100-kDa dynamin-related protein, we immunolocalized egg 100-kDa protein in cryosections and isolated cortices of unfertilized eggs. Egg 100-kDa protein was localized to the monolayer of cortical granules that underlie the plasma membrane and to the inner cytoplasm. Immunolocalizations of egg 100-kDa protein in cortices isolated from fertilized eggs detailed the redistribution of the egg 100-kDa onto cortically positioned vesicles. Egg 100-kDa dynamin-related protein was expressed throughout early development with approximately equivalent amounts present in the unfertilized egg and embryo through early gastrulation, followed by an abrupt decrease in the level of expression by the pluteus larval stage.
我们已启动一项研究，利用偶联到琼脂糖4B上的微管蛋白二聚体，从未受精的海胆卵中鉴定可能的微管蛋白二聚体结合蛋白。现已分离出一种100 kDa的多肽，并检测了其与微管蛋白/微管的相互作用。溶液组装研究表明，该100 kDa多肽可增强微管组装。该100 kDa蛋白还能与聚合微管聚集并共同沉降。兔抗卵100 kDa多克隆抗体与几种不同海胆物种卵中的一种100 kDa多肽以及与哺乳动物脑动力蛋白（一种100 kDa微管结合蛋白）发生交叉反应。这些生化特性和免疫学结果表明，海胆100 kDa微管结合蛋白似乎与动力蛋白家族有关。为深入了解海胆卵100 kDa动力蛋白相关蛋白的功能作用，我们在未受精卵的冷冻切片和分离的皮层中对卵100 kDa蛋白进行了免疫定位。卵100 kDa蛋白定位于质膜下方的皮层颗粒单层以及内部细胞质中。在从受精卵分离的皮层中对卵100 kDa蛋白进行免疫定位，详细显示了卵100 kDa蛋白重新分布到皮层定位的小泡上。卵100 kDa动力蛋白相关蛋白在整个早期发育过程中均有表达，在未受精卵和早期原肠胚形成前的胚胎中含量大致相当，随后在长腕幼虫阶段表达水平急剧下降。
