Immunoelectron microscopical analysis of intracellular processing of hormones by use of ultracryosection.

使用超冷冻切片对细胞内激素加工进行免疫电子显微镜分析。

基本信息

批准号：
02670004
负责人：
TANAKA Shigeyasu
金额：
$ 1.28万
依托单位：
Gunma University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1990
资助国家：
日本
起止时间：
1990 至 1991
项目状态：
已结题

项目摘要

To obtain antibodies specific for precursor pro-opiomelanocortiti(POMC)and its cleaved peptides, we prepared synthetic peptides corresponding to the cleavage site(ST-1)between ACTH and beta-lipotropic hormone moieties of POMC and to amidated joining peptide(JP ; ST-3)and generated their polyclonal antibodies in rabbits. The anti-ST-1 serum immunoprecipitated only POMC in the extract of AtT-20 cells. In addition to. a similar immunoprecipitation examination, immunoabsorption test showed that anti-ST-3 serum recognizes cleaved peptides, 16K amino-terminal peptide, and JP, but not POMC precursor. In ultracryosections of pituitary corticotrophs two distinct types of secretory granules were observed : one is small dense granules, and the other is large and lucent granules. The former was found not only near the Golgi apparatus, but also among the peripheral secretory granules arranged in a single row along the cell membrane. The latter was observed mainly among the peripheral secretory granules. Double-immunolabeling with gold particles of different sizes for the precursor POMC and its cleaved peptides revealed that the former was localized in electron-dense secretory granules, whereas the latter was found in the electron-lucent secretory granules in the same ultrathin cryosections. Colocalization of POMC and its cleaved peptides in the same secretory granules was rarely observed. Only POMC immunolabeling was detected in the Golgi cisternae. These results suggest that a certain step of proteolytic processing of POMC occurs in the secretory granules during the transition between the two distinct stages of secretory granule maturation in the rat anterior pituitary corticotroph.

为了获得前体阿片黑皮质素前体（POMC）及其裂解肽的特异性抗体，我们制备了与 POMC 的 ACTH 和 β-促脂激素部分之间的裂解位点（ST-1）相对应的合成肽和酰胺化连接肽（JP ; ST -3)并在兔子体内产生了多克隆抗体。抗ST-1血清仅免疫沉淀AtT-20细胞提取物中的POMC。此外。类似的免疫沉淀检查、免疫吸收试验显示抗ST-3血清识别裂解肽、16K氨基末端肽和JP，但不识别POMC前体。在垂体促肾上腺皮质激素的超冷冻切片中，观察到两种不同类型的分泌颗粒：一种是小致密颗粒，另一种是大而透明的颗粒。前者不仅存在于高尔基体附近，而且存在于沿细胞膜单行排列的外周分泌颗粒中。后者主要在外周分泌颗粒中观察到。用不同大小的金颗粒对前体 POMC 及其裂解肽进行双重免疫标记，结果显示前者位于电子致密的分泌颗粒中，而后者位于相同超薄冷冻切片中的电子透明的分泌颗粒中。很少观察到 POMC 及其裂解肽在同一分泌颗粒中的共定位。在高尔基池中仅检测到 POMC 免疫标记。这些结果表明，在大鼠垂体前叶促肾上腺皮质激素分泌颗粒成熟的两个不同阶段之间的过渡期间，POMC 的蛋白水解加工的某个步骤发生在分泌颗粒中。