Filson A J, Lewis A, Blobel G, Fisher P A
J Biol Chem. 1985 Mar 10;260(5):3164-72.
A high molecular weight glycoprotein found associated with a nuclear matrix-pore complex-lamina (NMPCL) preparation obtained from Drosophila melanogaster embryos has been shown by in vitro analyses to be largely confined to this subcellular fraction. In contrast with several of the NMPCL proteins, this glycoprotein remains completely insoluble after treatment with 5 M urea. It has, therefore, been possible to separate the glycoprotein from other NMPCL components by differential urea extraction. The glycoprotein in the 5 M urea-extracted pellet has been solubilized by boiling in sodium dodecyl sulfate and purified to near-homogeneity by sequential steps of chromatography on hydroxylapatite and Sephacryl S-300 (both run in the presence of 0.1% sodium dodecyl sulfate), followed by affinity chromatography on lentil lectin-Sepharose. Over 30 hybridoma cell lines producing antibodies against this glycoprotein have been obtained. Monoclonality has been established for two of these lines (designated AGP-26 and AGP-78), and the antibodies they secrete have been further characterized. Western blot analysis has shown both antibodies to be monospecific (with respect to other Drosophila embryo polypeptides) for the major NMPCL glycoprotein; in addition, antibody AGP-78 has been shown to be weakly cross-reactive with glycoproteins of similar or identical molecular weight found associated with isolated nuclear fractions obtained from Xenopus oocytes, as well as chicken, opossum, and rat livers. Finally, both antibodies AGP-26 and AGP-78 react exclusively with the Drosophila nuclear periphery (nuclear envelope) in situ as demonstrated by indirect immunofluorescence analysis of larval cryosections. Based on these results as well as upon those of biochemical studies reported previously (Berrios, M., Filson, A. J., Blobel, G, and Fisher, P. A. (1983) J. Biol. Chem. 258, 13384-13390), we conclude that the major Drosophila NMPCL glycoprotein is the specific homolog of the high molecular weight glycoprotein recently shown using immunoelectron microscopy to be a distinct component of the rat liver nuclear pore complex (Gerace, L., Ottaviano, Y., and Kondor-Koch, C. (1982) J. Cell Biol. 95, 826-837).
从黑腹果蝇胚胎中获得的一种与核基质 - 核孔复合体 - 核纤层（NMPCL）制剂相关的高分子量糖蛋白，经体外分析表明，它主要局限于这一亚细胞组分。与几种NMPCL蛋白不同，这种糖蛋白在用5M尿素处理后仍完全不溶。因此，通过差示尿素提取法可将该糖蛋白与其他NMPCL组分分离。5M尿素提取沉淀中的糖蛋白经十二烷基硫酸钠煮沸后溶解，并通过在羟基磷灰石和Sephacryl S - 300上的连续层析步骤（均在0.1%十二烷基硫酸钠存在下进行），然后在扁豆凝集素 - 琼脂糖上进行亲和层析，纯化至近乎均一。已获得30多个产生针对这种糖蛋白抗体的杂交瘤细胞系。已确定其中两个细胞系（命名为AGP - 26和AGP - 78）的单克隆性，并对它们分泌的抗体进行了进一步表征。蛋白质印迹分析表明，两种抗体对主要的NMPCL糖蛋白都是单特异性的（相对于其他果蝇胚胎多肽）；此外，已表明抗体AGP - 78与从非洲爪蟾卵母细胞以及鸡、负鼠和大鼠肝脏分离的核组分中发现的分子量相似或相同的糖蛋白有弱交叉反应。最后，通过幼虫冰冻切片的间接免疫荧光分析表明，抗体AGP - 26和AGP - 78在原位仅与果蝇核周边（核膜）反应。基于这些结果以及先前报道的生化研究结果（贝里奥斯，M.，菲尔森，A. J.，布洛贝尔，G，和费舍尔，P. A.（1983年）《生物化学杂志》258，13384 - 13390），我们得出结论，主要的果蝇NMPCL糖蛋白是最近使用免疫电子显微镜显示为大鼠肝细胞核孔复合体独特组分的高分子量糖蛋白的特异性同源物（杰拉斯，L.，奥塔维亚诺，Y.，和孔多尔 - 科赫，C.（1982年）《细胞生物学杂志》95，826 - 837）。
