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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.

为了获得对前体阿黑皮素原（POMC）及其裂解肽特异的抗体，我们制备了对应于POMC的ACTH和β-促脂激素部分之间的裂解位点（ST-1）和酰胺化连接肽（JP ; ST-3）的合成肽，并在兔中产生了它们的多克隆抗体。抗ST-1血清仅免疫沉淀AtT-20细胞提取物中的POMC。除了.类似的免疫沉淀试验、免疫吸附试验显示，抗ST-3血清识别裂解肽、16 K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细胞的冰冻切片中，观察到两种不同类型的分泌颗粒：一种是小而致密的颗粒，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沿着排列成一排的分泌颗粒周围。后者主要见于外周分泌颗粒。用不同大小的金颗粒对前体POMC及其裂解肽进行双免疫标记，发现前者定位于电子致密的分泌颗粒中，而后者则在相同的冰冻切片中发现于电子透明的分泌颗粒中。POMC及其裂解肽在同一分泌颗粒中的共定位很少观察到。在高尔基体中仅检测到POMC免疫标记。这些结果表明，POMC的蛋白水解加工的某个步骤发生在分泌颗粒之间的两个不同阶段的大鼠垂体前叶促肾上腺皮质激素分泌颗粒成熟的过渡。