Variation and immunocyochemical localization of processing enzymes in proopiomelanocortin cells during ontoneny

原阿黑皮素细胞在色调过程中加工酶的变化和免疫细胞化学定位

基本信息

批准号：
04670002
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：
1992
资助国家：
日本
起止时间：
1992 至 1993
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-04670002/
关键词：
Processing Processing enzyme Secretory granule Pituitary gland Immunocyochemistry 内分泌細胞

项目摘要

PC1/PC3 and PC2 are endoproteases involved in prohormone cleavage at pairs of basic amino acids. To determine the cellular and subcellular distribution of PC1/PC3 and PC2 in the pituitary glands, we generated their polyclonal antisera in rabbits, using two synthetic peptide antigens corresponding to amino acids 442-459 (ST-28) of PC1/PC3 and 613-629 (ST-29) of PC2 and two bacterially expressed antigens covering amino acids 145-414 (KN-1) of PC1/PC3 and 385-637 (KN-2) of PC2. Western blot analysis revealed the presence of PC1/PC3 87 and 68 kDa) and PC2 (70 kDa) in the rat pancreatic islets, indicating that each of the antisera was specific for the corresponding antigen. In the present immunocyochemical studies, many neuroendcrine cells displayd positive reaction to these antibodies. In almost all the animals as far as examined, PC1/PC3 and PC2 showed differential immunoreactivity in proopiomelanocortin (POMC) cells. In the anterior lobe, antiserum against PC1/PC3 exclusively immunostained corticotrophs in the intermediate lobe. Immunoelectron microscopy, using double-labeling with gold particles of different sizes revealed that almost all of immunolabeled ACTH- or alpha-MSH-secretory granules were also positive to PC1/PC3 and PC2 antibody. This differential localization of PC1/PC3 and PC2 provides a novel argument for the subtrate-specificity of the PC1/PC3 and PC2 enzymes in the processing of pituitary POMC cells. To elucidate factors that regulate changes in the processing patterns of POMC in the pituitary corticotrophs during postnatal development, in near further, the presence of PC1/PC3 and PC2 will be examined immunocytochemically.

PC1/PC3 和 PC2 是参与碱性氨基酸对的激素原裂解的内切蛋白酶。为了确定 PC1/PC3 和 PC2 在垂体中的细胞和亚细胞分布，我们使用对应于 PC1/PC3 和 613-氨基酸 442-459 (ST-28) 的两种合成肽抗原，在兔子中产生了它们的多克隆抗血清。 PC2 的 629 (ST-29) 和覆盖 PC1/PC3 的氨基酸 145-414 (KN-1) 的两种细菌表达的抗原和 PC2 的 385-637 (KN-2)。蛋白质印迹分析显示大鼠胰岛中存在 PC1/PC3 87 和 68 kDa) 和 PC2 (70 kDa)，表明每种抗血清对相应抗原具有特异性。在目前的免疫细胞化学研究中，许多神经内分泌细胞对这些抗体表现出阳性反应。就所检查的几乎所有动物而言，PC1/PC3 和 PC2 在阿黑皮素原 (POMC) 细胞中表现出不同的免疫反应性。在前叶中，抗 PC1/PC3 的抗血清专门免疫染色中叶中的促肾上腺皮质激素。使用不同大小的金颗粒双标记的免疫电子显微镜显示，几乎所有免疫标记的 ACTH 或 α-MSH 分泌颗粒也对 PC1/PC3 和 PC2 抗体呈阳性。 PC1/PC3 和 PC2 的这种差异定位为 PC1/PC3 和 PC2 酶在垂体 POMC 细胞加工中的底物特异性提供了新的论据。为了阐明出生后发育过程中调节垂体促肾上腺皮质激素中 POMC 加工模式变化的因素，近一步，将通过免疫细胞化学方法检查 PC1/PC3 和 PC2 的存在。