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.

为了获得特异性的抗体前甲状腺皮质皮质（POMC）及其裂解的肽，我们制备了与ACTH和β-甲状腺激素之间的裂解位点（ST-1）相对应的合成肽，POMC和POMC的β-甲状腺激素部分是POMC和amagied obsing obsing肽（Jp; jp; st-3）的polotic and prodip in polycl of in capec; st-clodec int colder int clodect in polodcl of他们的polyclon。抗ST-1血清免疫沉淀仅在ATT-20细胞提取物中POMC。此外。类似的免疫沉淀检查，免疫吸收测试表明，抗ST-3血清识别裂解的肽，16K氨基末端肽和JP，但不识别POMC前体。在垂体皮质营养的超子中，观察到两种不同类型的分泌颗粒：一种是小颗粒，另一个是大的颗粒。前者不仅在高尔基体附近，而且在沿细胞膜沿单一行排列的外围分泌颗粒中发现。后者主要观察到外围分泌颗粒中。前体POMC及其切割肽的双颗粒具有不同尺寸的金颗粒，显示前者位于电子密集的分泌颗粒中，而后者则在同一超级冻结的电子灯液分泌颗粒中发现了后者。很少观察到POMC及其在同一分泌颗粒中裂解的肽的共定位。在高尔基水库中仅检测到POMC免疫标记。这些结果表明，在大鼠前垂体皮质营养的大鼠前分泌颗粒成熟的两个不同阶段之间的过渡期间，pOMC的蛋白水解处理的一定步骤发生在分泌颗粒中。