Immunocytochemical study of prorenin procrssing in the mouse submandibular gland, using antibodies against prorenin and its cleaved fragments.

使用针对肾素原及其切割片段的抗体对小鼠颌下腺中肾素原加工进行免疫细胞化学研究。

基本信息

批准号：
14571753
负责人：
KURABUCHI Shingo
金额：
$ 1.86万
依托单位：
The Nippon Dental University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2002
资助国家：
日本
起止时间：
2002 至 2003
项目状态：
已结题

项目摘要

According to the complete amino acid sequence of the mouse submandibular gland renin precursor (Ren-2-prorenin) (Panthier et al.,1982; Misono et al.,1982), the short peptides, which stride the positions of proteic cleavages, each occuring after three basic residues : Cys 18-Thr 19, Mg 63-Der 64, and Mg 363-Asp 364, respectively, were designed, chemically synthesized, and immunized rabbits. The preparation of specific antibody against the second peptide, ST-161: Thr-Lys-Arg-Ser-Ser-Leu-Thr-Asp-Leu-Ile-Cys, which strides between the Lys62-Arg63 was succeeded. This antibody seemes to recognize prorenin, but not mature renin. Western blotting analysis, however, was not performed to examin the specificity of the antibody. It was unluckly that other peptides designed by the author were not reacted as the antigen, so, the antibodies were not obtained.The immunocytochemical localization and distribution of the ST-161 was examined in the mouse submandibular gland by indirect Cy3 (indocarbocyanine)-labeled antibody method for light microscopy. Red immunoreactive materrials were localized at the pen-nuclear region of the granular convoluted tubule cells, but not on the secretory granules. These immunostain-ed region was corresponding to the trans-Golgi network, containing Golgi apparatus and immature secretory granules. It is therefore concluded that the corresponding portion of mouse Ren-2-prorenin may be cleaved off in the Golgi apparatus or immumature secretory granules. It has not been successful to date to confirm this idea by immunoelectron microscopy.

根据小鼠颌下腺肾素前体（Ren-2-prorenin）的完整氨基酸序列（Panthier et al.,1982; Misono et al.,1982），跨越蛋白质裂解位置的短肽，每个发生在三个碱性残基之后：Cys 18-Thr 19、Mg 63-Der 64 和 Mg分别设计、化学合成并免疫兔子363-Asp 364。成功制备了针对跨越Lys62-Arg63之间的第二肽ST-161：Thr-Lys-Arg-Ser-Ser-Leu-Thr-Asp-Leu-Ile-Cys的特异性抗体。该抗体似乎识别肾素原，但不识别成熟肾素。然而，没有进行蛋白质印迹分析来检查抗体的特异性。不幸的是，作者设计的其他肽没有作为抗原发生反应，因此，没有获得抗体。通过间接Cy3（吲哚羰花青）标记，检查了ST-161在小鼠颌下腺中的免疫细胞化学定位和分布。用于光学显微镜的抗体方法。红色免疫反应物质位于颗粒曲管细胞的笔核区域，但不在分泌颗粒上。这些免疫染色区域对应于跨高尔基体网络，包含高尔基体和未成熟的分泌颗粒。因此得出结论，小鼠Ren-2-肾素原的相应部分可能在高尔基体或未成熟的分泌颗粒中被裂解掉。迄今为止，尚未成功通过免疫电子显微镜证实这一想法。