Topological distribution of two connexin32 antigenic sites in intact and split rodent hepatocyte gap junctions
Topological distribution of two connexin32 antigenic sites in intact and split rodent hepatocyte gap junctions
复制标题
完整和分裂啮齿动物肝细胞间隙连接中两个 connexin32 抗原位点的拓扑分布
DOI：
--
复制
发表时间：
1988
影响因子：
7.8
通讯作者：
Lynne Jesaitis
中科院分区：
文献类型：
--
作者：
D. Goodenough;David L. Paul;Lynne Jesaitis
connexin32 是多种细胞类型间隙连接的主要多肽，已使用针对与从 cDNA 克隆推导的氨基酸序列相对应的合成寡肽产生的位点特异性抗血清，在大鼠和小鼠肝细胞间隙连接中研究了 connexin32 的膜拓扑结构。根据已发表的亲水性图谱和确定的蛋白酶敏感切割位点，合成了与 connexin32 的两个亲水结构域相对应的寡肽，一个预计面向细胞质，另一个预计面向细胞外。将抗血清培养到寡肽的匙孔血蓝蛋白缀合物上，并使用间接免疫细胞化学在分离的间隙连接上绘制其抗原的分布图。结果直接证明了 connexin32 序列的氨基酸 98-124 中包含的抗原的细胞质取向。完整、分离的间隙连接中的细胞外空间太小，无法结合抗体分子，因此需要使用尿素/碱程序对连接膜进行实验分离以暴露其细胞外表面。虽然氨基酸 164-189 中包含的抗原在一些分离的连接膜的细胞外表面上可见，但观察结果和分裂程序的变异性在分离连接膜所需的变性条件后留下了关于观察结果的生物学相关性的模糊性。然而，使用不同的方法，可以使用高渗二糖连接分裂程序将抗原暴露在完整的肝脏中。细胞表面抗原暴露的时间似乎在 30 秒时达到峰值，并在 2-4 分钟后消失。总而言之，这些数据证明了氨基酸 164-189 内所含抗原的细胞外取向，这可能涉及间隙连接内的细胞间相互作用。
The membrane topology of connexin32, a principal polypeptide of gap junctions in diverse cell types, has been studied in rat and mouse hepatocyte gap junctions using site-specific antisera raised against synthetic oligopeptides corresponding to amino acid sequences deduced from cDNA clones. Based on published hydropathicity maps and identified protease-sensitive cleavage sites, oligopeptides were synthesized corresponding to two hydrophilic domains of connexin32, one predicted to face the cytoplasm, the other predicted to be directed extracellularly. Antisera were raised to keyhole limpet hemocyanin conjugates of the oligopeptides and used to map the distribution of their antigens using indirect immunocytochemistry on isolated gap junctions. The results directly demonstrated the cytoplasmic orientation of an antigen contained within amino acids 98-124 of the connexin32 sequence. The extracellular space in intact, isolated gap junctions is too small to permit binding of antibody molecules, necessitating the experimental separation of the junctional membranes to expose their extracellular surfaces using a urea/alkali procedure. While an antigen contained within amino acids 164-189 was visualized on the extracellular surfaces of some of the separated junctional membranes, variability in the observations and in the splitting procedure left ambiguities concerning the biological relevance of the observations after the denaturing conditions necessary to separate the junctional membranes. Using a different approach, however, the antigen could be exposed in intact liver using a hypertonic disaccharide junction-splitting procedure. The period of time of antigen exposure at the cell surface appears to peak at 30 s and disappear by 2-4 min. Taken together, these data demonstrate the extracellular orientation of an antigen contained within amino acids 164-189, which may be involved in cell-cell interaction within the gap junction.
影响因子：
5.6
作者：
Makowski,L;Caspar,DL;Phillips,WC;Goodenough,DA
通讯作者：
Goodenough,DA
对分离的完整大鼠肝脏间隙连接和间隙连接衍生的单膜结构中主要蛋白质的拓扑分析。
DOI：
--
发表时间：
1987
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Zimmer,DB;Green,CR;Evans,WH;Gilula,NB
通讯作者：
Gilula,NB