Ultrastructural localization of insulin and C-peptide antigenic sites in rat pancreatic B cell obtained by applying the quantitative high-resolution protein A-gold approach.
Ultrastructural localization of insulin and C-peptide antigenic sites in rat pancreatic B cell obtained by applying the quantitative high-resolution protein A-gold approach.
复制标题
应用定量高分辨率蛋白 A-gold 方法获得大鼠胰腺 B 细胞中胰岛素和 C 肽抗原位点的超微结构定位。
DOI：
10.1002/aja.1001850213
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发表时间：
1989
影响因子：
--
通讯作者：
M. Bendayan
中科院分区：
文献类型：
--
作者：
M. Bendayan
应用免疫组织化学和细胞化学技术，在大鼠胰腺B细胞上发现了胰岛素和C-肽的抗原部位。在光学显微镜水平上的荧光素和罗丹明染色在同一B细胞中检测到这两种抗原。用蛋白A-金标记法，在粗面内质网池、移行元件池、高尔基体除最外的所有池、光滑但不包被的囊泡、未成熟和成熟的分泌颗粒以及一些溶酶体(多颗粒)结构中均可见这两种抗原的标记。所采用的固定方法获得了良好的超微结构保存，从而实现了高分辨率。各种对照实验表明，结果具有较高的特异性。定量评估证实了定性观察，因为他们记录了标记的特异性，并揭示了沿着内质网-高尔基-颗粒分泌途径的两个抗原位置存在递增的梯度。定量结果还显示了出现标记增加的不同部位：粗面内质网、光滑小泡、高尔基体的跨池以及未成熟和成熟的分泌颗粒。高尔基体由三个不同的小室组成，根据它们的标记浓度进行区分。这些脑池包括顺侧的池、横侧的池和最坚硬的池。由于胰岛素和C-肽形成胰岛素原链，所以它们的抗原部位位于分泌途径的相同位置；位置的差异只出现在分泌颗粒中，胰岛素集中在分泌颗粒的核心，而C-肽则同时存在于分泌颗粒的核心和晕环中。此外，在显示结晶核心的成熟分泌颗粒中，胰岛素被限制在核心，而C肽被限制在晕层。这些结果与生化数据一致，表明这两个抗原位点同时定位在未成熟分泌颗粒上游的隔室，反映了它们以胰岛素原的形式存在。然而，当胰岛素原解离为胰岛素和C-肽时，这两个抗原点被分离在不同的位置。这些多肽似乎共享平行的途径和命运，包括通过胞吞作用或通过溶酶体系统降解的分泌。
Insulin and C-peptide antigenic sites have been revealed in rat pancreatic B cells by applying immunohistochemical and cytochemical techniques. Fluorescein and rhodamine stains at the light-microscope level have detected both antigens in the same B cells. With the protein A-gold technique, labeling for both antigens was found in the cisternae of the rough endoplasmic reticulum, in those of the transitional elements, in all the cisternae of the Golgi apparatus except in the trans-most one, in the smooth but not in the coated vesicles, in the immature and mature secretory granules, and in some lysosomal (multigranular) structures. The fixation procedure used yielded excellent ultrastructural preservation which allowed for high resolution. The various control experiments demonstrated the high specificity of the results. Quantitative evaluations confirmed the qualitative observations in that they documented the specificity of the label and revealed the presence of an increasing gradient for both antigenic sites along the endoplasmic reticulum-Golgi-granule secretory pathway. The quantification also demonstrated various sites in which an increased labeling occurs: the rough endoplasmic reticulum, the smooth vesicles, the trans-cisternae of the Golgi apparatus, and the immature and the mature secretory granules. The Golgi apparatus was composed of three different subcompartments distinguished by their concentration of label. These include the cisternae on the cis-side, those on the trans-side, and the trans-most rigid cisternae. Since insulin and C-peptide form the proinsulin chain, their antigenic sites were found in the same locations along the secretory pathway; differences in location appeared only in the secretory granules, where insulin was concentrated in the core, while C-peptide was found in both the core and the halo of the granules. Furthermore, in the mature secretory granules displaying a crystalline core, insulin was restricted to the core, while C-peptide was confined to the halo. These results are in accord with the biochemical data, which indicate that simultaneous localization of both antigenic sites in compartments upstream to the immature secretory granules reflects their presence in the form of proinsulin. However, upon dissociation of proinsulin into insulin and C-peptide, both antigenic sites are segregated in different locations. The peptides appear to share parallel pathways and a fate which includes secretion through exocytosis or degradation by the lysosomal system.