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Structural Studies of Tissue Specificity and Diversity of Gap Junctions.

组织特异性和间隙连接多样性的结构研究。

基本信息

批准号：
03454117
负责人：
SHIBATA Yosaburo
金额：
$ 3.65万
依托单位：
KYUSHU UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1991
资助国家：
日本
起止时间：
1991 至 1992
项目状态：
已结题

项目摘要

Molecular architectures of Liver Gap Junctions: The localizations of gap junction proteins, Cx32 and Cx26 were examined in rat and guinea pig livers by using site-specific antibodies against synthetic oligopeptides. Double labeling immunofluorescence microscopy revealed that in guinea pig liver both proteins spread throughout the liver lobules and seemed to localize together within the same gap junctional plaque. Quick-freeze, deep-etch immunoelectron microscopy showed that immunolabeling of isolated guinea pig liver gap junctional plaques with either Cx32 or Cx26 antiserum yielded complete and dense antibody decoration of the cytoplasmic surface of the plaques. We did not observe any focal or patchy clusters of the labeling in any plaques examined. Double labeling immuno electron microscopy confirmed that both Cx32 and Cx26 are colocalized in the same gap junction plaques. These results suggest that in the hepatocytes expressing both Cx32 and Cx26, both types of gap junction proteins are not segregated but intermix randomly within the same plaques.Cytoplasmic Surface Structures of Bovine lens Fiber Gap Junctions: Bovine lens fibers studied by quick freeze-deep etch replica method revealed particulate substructures on cytoplasmic surfaces in situ. In isolated membranes, thin section electron microscopy showed that 16-17 nm pentalamellar junctions were included in this specimen. Deep-etch replica showed that gap junctional plaques also had particles on their cytoplasmic surfaces clearly distinguished from the surrounding particle-free non-gap junctional surfaces. Treatment of isolated lens membranes with endoproteinase glu-C induced cleavage of MP70 to MP38 in SDS-PAGE and loss of their antigenicity to the anti-MP70 mono-clonal antibody, whereas the particulate patterns persisted still on the cytoplasmic surfaces of gap junctional plaques, suggesting the coexistance of additional proteins other than MP70 in the lens fiber gap junction structures.

肝间隙连接的分子结构：利用针对合成寡肽的位点特异性抗体，在大鼠和豚鼠肝脏中检测了间隙连接蛋白Cx32和Cx26的定位。双标记免疫荧光显微镜显示，在豚鼠肝脏中，这两种蛋白遍布整个肝小叶，似乎在同一间隙连接斑块内共同定位。速冻、深蚀刻免疫电镜显示，用Cx32或Cx26抗血清对分离的豚鼠肝间隙连接斑块进行免疫标记，可在斑块的细胞质表面产生完整而致密的抗体装饰。在检查的任何斑块中，我们没有观察到任何局灶性或斑片状的标记簇。双标记免疫电镜证实，Cx32和Cx26共定位于相同的间隙连接斑块。这些结果表明，在同时表达Cx32和Cx26的肝细胞中，两种类型的间隙连接蛋白不是分离的，而是在同一斑块内随机混合。牛晶状体纤维间隙连接的细胞质表面结构：用快速冷冻深蚀刻复制方法研究牛晶状体纤维，在细胞质表面原位显示颗粒亚结构。在离体膜上，薄层电镜显示该样品包含16-17 nm的五层结。深蚀刻复制显示，间隙连接斑块的细胞质表面也有颗粒，与周围无颗粒的非间隙连接表面明显不同。用内源性蛋白酶glue -c处理离体晶状体膜，诱导SDS-PAGE中MP70裂解为MP38，其对抗MP70单克隆抗体的抗原性丧失，而间隙连接斑块的胞质表面仍然存在颗粒模式，这表明晶状体纤维间隙连接结构中存在除MP70以外的其他蛋白质。