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

为了获得针对前体阿片黑质皮质炎(POMC)及其裂解肽的抗体，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)和酰胺化连接肽(JP；ST-3)相对应的合成肽，并在兔体内制备了它们的多克隆抗体。抗ST-1血清免疫沉淀在AtT-20细胞提取液中仅表达POMC。除了……之外。免疫沉淀试验、免疫吸收试验表明，抗ST-3血清可识别裂解肽、16K氨基末端多肽和JP，但不识别POMC前体。在垂体促肾上腺皮质激素细胞的超低温切片中，观察到两种不同类型的分泌颗粒：一种是小而致密的颗粒，另一种是大而透亮的颗粒。前者不仅存在于高尔基体附近，而且存在于沿细胞膜呈单列排列的外周分泌颗粒中。后者主要见于外周分泌颗粒。用不同大小的金粒子对前体POMC及其裂解肽进行双重免疫标记，发现前者定位于电子密度较高的分泌颗粒中，而后者定位于同一超薄切片中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共定位。高尔基体池仅检测到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过程中，POMC的蛋白降解过程发生在分泌颗粒中。

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.