Pudles J, Moudjou M, Hisanaga S, Maruyama K, Sakai H
CNRS URA 1131, Université de Paris-Sud, Orsay, France.
Exp Cell Res. 1990 Aug;189(2):253-60. doi: 10.1016/0014-4827(90)90244-5.
A giant protein of apparent molecular weight (Mr) 2000 kDa, as determined by SDS-PAGE, was isolated and partially purified, under denaturing conditions, from the detergent-resistant cytomatrix of unfertilized sea urchin egg. Immunoblot analysis and indirect immunofluorescence microscopy observations indicated that this high-molecular-weight protein cross-reacted with the immunospecific serum raised against chicken breast muscle beta-connectin. However, rotary-shadowing electron microscopy images of the protein revealed short threadlike structures which appear morphologically different from beta-connectin structure. Indirect immunofluorescence localization of the protein with anti-beta-connectin serum showed a distribution throughout the whole unfertilized egg cytomatrix. This immunofluorescence pattern seems to change upon egg fertilization, since at metaphase the fluorescence stain appears to be excluded from the mitotic apparatus region as revealed by the double immunolabeling with anti-beta-connectin serum and monoclonal anti-alpha-tubulin antibody. Moreover, when egg cortical fragments were double-labeled with anti-beta-connectin serum and rhodamin-conjugated phalloidin, it was observed that the microfilaments assembled after fertilization seem to be in close association with the protein at the cleavage furrow and other locations. The possible significance of this sea urchin egg connectin(titin)-like protein is discussed.
通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）测定，从未受精海胆卵的抗去污剂细胞基质中，在变性条件下分离并部分纯化出一种表观分子量（Mr）为2000 kDa的巨大蛋白质。免疫印迹分析和间接免疫荧光显微镜观察表明，这种高分子量蛋白质与针对鸡胸肌β-连接蛋白产生的免疫特异性血清发生交叉反应。然而，该蛋白质的旋转阴影电子显微镜图像显示出短的丝状结构，其形态与β-连接蛋白结构不同。用抗β-连接蛋白血清对该蛋白质进行间接免疫荧光定位，结果显示其分布于整个未受精卵细胞基质中。这种免疫荧光模式在卵子受精后似乎会发生变化，因为在中期，通过抗β-连接蛋白血清和单克隆抗α-微管蛋白抗体的双重免疫标记显示，荧光染色似乎被排除在有丝分裂器区域之外。此外，当用抗β-连接蛋白血清和罗丹明偶联的鬼笔环肽对卵皮质片段进行双重标记时，观察到受精后组装的微丝似乎在卵裂沟和其他位置与该蛋白质紧密相关。本文讨论了这种海胆卵连接蛋白（肌联蛋白）样蛋白质的可能意义。
