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

PC1/PC3和PC2是参与激素原切割的碱性氨基酸对的内源性蛋白酶。为了确定PC1/PC3和PC2在兔垂体中的细胞和亚细胞分布，我们合成了PC1/PC3的442-459 （ST-28）和PC2的613-629 （ST-29）氨基酸对应的两种肽抗原，以及两种细菌表达的PC1/PC3的145-414 （KN-1）和PC2的385-637 （KN-2）氨基酸抗原，制备了它们的多克隆抗血清。Western blot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。在几乎所有被检测的动物中，PC1/PC3和PC2在POMC细胞中表现出不同的免疫反应性。在前叶，在中间叶，抗PC1/PC3特异性免疫染色的促皮质细胞的抗血清。免疫电镜下，用不同大小的金颗粒进行双标记，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的差异定位为垂体POMC细胞加工过程中PC1/PC3和PC2酶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皮质细胞POMC加工模式变化的因素，在不久的将来，我们将通过免疫细胞化学检测PC1/PC3和PC2的存在。

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.