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

为了获得针对前体POMC （proopiomelanocortiti）及其裂解肽的抗体，我们合成了POMC ACTH与β -增脂激素部分之间的裂解位点ST-1和修饰的连接肽JP ST-3对应的肽段，并在家兔体内制备了它们的多克隆抗体。抗st -1血清仅免疫沉淀at -20细胞提取物中的POMC。除了…类似的免疫沉淀检查、免疫吸收试验显示抗st -3血清可识别裂解肽、16K氨基末端肽和JP，但不能识别POMC前体。垂体促肾上腺皮质激素的超微切片显示两种不同类型的分泌颗粒：一种是小而致密的颗粒，另一种是大而明亮的颗粒。前者不仅存在于高尔基体附近，也存在于沿细胞膜排列成单排的外周分泌颗粒中。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，而后者则定位于相同超薄切片的电子透明的分泌颗粒中。在相同的分泌颗粒中很少观察到POMC及其裂解肽的共定位。高尔基池中仅检测到POMC免疫标记。这些结果表明，在大鼠垂体前皮质细胞分泌颗粒成熟的两个不同阶段之间的过渡过程中，POMC的蛋白水解过程在分泌颗粒中发生了一定的步骤。

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.