Monoclonal antibodies identify two novel proteins associated with vasopressin secretory granules of the rat neurohypophysis.
Monoclonal antibodies identify two novel proteins associated with vasopressin secretory granules of the rat neurohypophysis.
复制标题
单克隆抗体鉴定出两种与大鼠神经垂体加压素分泌颗粒相关的新蛋白质。
DOI：
10.1016/0006-8993(91)91281-5
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发表时间：
1991
期刊：
影响因子：
2.9
通讯作者：
Hapner,SJ
中科院分区：
文献类型：
--
作者：
Paden,CM;Hapner,SJ
免疫细胞化学和免疫印迹技术已用于表征两种单克隆抗体（MAb C6 和 D5）所识别的抗原，该抗体是针对新生大鼠视上（SON）和室旁（PVN）下丘脑核的分离细胞产生的。过氧化物酶免疫细胞化学显示，两种 MAb 均标记成人和新生儿 SON 和 PVN 中的大细胞核周以及视交叉上核中的较小神经元。下丘脑-神经垂体束的轴突也在下丘脑和正中隆起的内带内进行免疫标记，C6 和 D5 各自与成人和新生儿神经垂体特异性结合。使用 C6 或 D5 同时与针对催产素、神经素或加压素神经素特异性的兔抗血清进行双标记免疫荧光实验表明，C6 仅与 SON 中的加压素能大细胞周核细胞结合，而 D5 既标记加压素能神经元，又标记一小部分催产素能大细胞神经元。在超微结构水平上对 MAb 与神经垂体结合的包埋后免疫金分析表明，C6 和 D5 均识别与神经分泌轴突子集中的大致密核心神经分泌颗粒相关的抗原。使用 SDS-PAGE 和蛋白质免疫印迹对 C6 和 D5 识别的抗原进行初步生化表征。 MAb C6 和 D5 在成人神经中间叶还原提取物的印迹中分别标记表观分子量为 38 和 68 kDa 的单个蛋白条带。 C6 和 D5 与神经生理素之间没有明显的交叉反应，抗神经生理素血清也不能识别由 C6 和 D5 识别的条带。因此，我们将这些新抗原指定为加压素颗粒蛋白的 VPGP38 和 VPGP68。
Immunocytochemical and immunoblotting techniques have been used to characterize the antigens recognized by two monoclonal antibodies (MAbs C6 and D5) produced against dissociated cells from punches of neonatal supraoptic (SON) and paraventricular (PVN) hypothalamic nuclei of the rat. Peroxidase immunocytochemistry revealed that both MAbs label magnocellular perikarya in the adult and neonatal SON and PVN as well as smaller neurons in the suprachiasmatic nucleus. Axons of the hypothalamo-neurohypophysial tract are also immunolabeled within the hypothalamus and zona interna of the median eminence, and C6 and D5 each bind specifically to both the adult and neonatal neurohypophysis. Dual-label immunofluorescence experiments employing C6 or D5 simultaneously with rabbit antisera specific for either oxytoxin, neurophysin or vasopressin neurophysin revealed that C6 binds only to vasopressinergic magnocellular perikarya in the SON, while D5 labels both vasopressinergic and a small subset of oxytocinergic magnocellular neurons. Post-embedding immunogold analysis of MAb binding to the neurohypophysis at the ultrastructural level showed that both C6 and D5 recognize antigens associated with large dense core neurosecretory granules in a subset of neurosecretory axons. Initial biochemical characterization of the antigens recognized by C6 and D5 was performed using SDS-PAGE and Western immunoblotting. MAbs C6 and D5 label single protein bands with apparent molecular weights of 38 and 68 kDa, resp., in blots of reduced extracts from the adult neurointermediate lobe. No cross-reactivity between C6 and D5 and the neurophysins was apparent, nor did anti-neurophysin sera recognize the bands identified by C6 and D5. We have therefore designated these novel antigens as VPGP38 and VPGP68 for VasoPressin Granule Proteins.