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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.

PC 1/PC 3和PC 2是参与在成对碱性氨基酸处的激素原切割的内切蛋白酶。为了确定PC 1/PC 3和PC 2在垂体中的细胞和亚细胞分布，我们制备了它们的多克隆抗血清，使用对应于PC 1/PC 3的氨基酸442-459（ST-28）和PC 2的氨基酸613-629（ST-29）的两种合成肽抗原和覆盖PC 1/PC 3的氨基酸145-414（KN-1）的两种细菌表达抗原，PC 3和PC 2的385-637（KN-2）。Western印迹分析显示大鼠胰岛中存在PC 1/PC 3（87和68 kDa）和PC 2（70 kDa），表明每种抗血清对相应的抗原具有特异性。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呈阳性反应。在几乎所有的动物中，PC 1/PC 3和PC 2在前阿黑皮素（POMC）细胞中表现出不同的免疫反应性。在前叶，抗血清对PC 1/PC 3专门免疫染色的促肾上腺皮质激素在中叶。免疫电子显微镜，使用不同大小的金颗粒的双标记显示，几乎所有的免疫标记的ACTH或α-MSH分泌颗粒也阳性的PC 1/PC 3和PC 2抗体。PC 1/PC 3和PC 2的这种差异定位为PC 1/PC 3和PC 2酶在垂体POMC细胞加工中的底物特异性提供了新的论据。为了阐明在出生后发育过程中调节垂体促肾上腺皮质激素细胞中POMC加工模式变化的因素，在不久的将来，将对PC 1/PC 3和PC 2的存在进行免疫细胞化学检查。