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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.

根据小鼠下颌下腺肾素前体（Ren-2-原肾素）的完整氨基酸序列（Panthier等人，1982; Misono等人，1982）中，设计、化学合成了跨越蛋白质切割位置的短肽，每个短肽分别出现在三个碱性残基之后：Cys 18-Thr 19、Mg 63-Der 64和Mg 363-Asp 364，并免疫家兔。成功制备了ST-161：Thr-Lys-Arg-Ser-Ser-Leu-Thr-Asp-Leu-Ile-Cys的特异性抗体。该抗体可识别原肾素，但不识别成熟的肾素。然而，没有进行蛋白质印迹分析来检查抗体的特异性。本实验采用Cy 3（indocarbocyanine）间接标记抗体的方法，在光镜下观察了ST-161在小鼠下颌下腺的免疫细胞化学定位和分布。在颗粒曲小管细胞的笔核区有红色免疫反应物质，而在分泌颗粒上没有。这些免疫染色区域与高尔基体网络相对应，含有高尔基体和未成熟的分泌颗粒。因此，小鼠Ren-2-prenin的相应部分可能在高尔基体或免疫分泌颗粒中被切割掉。迄今为止，通过免疫电子显微镜证实这一想法还不成功。