Structural Studies of Tissue Specificity and Diversity of Gap Junctions.

肝脏间隙连接的分子结构：通过使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查间隙连接蛋白Cx 32和Cx 26的定位。双标记免疫荧光显微镜显示，在豚鼠肝脏中，这两种蛋白质分布在整个肝小叶，似乎本地化在同一间隙连接斑块在一起。速冻，深蚀刻免疫电镜显示，免疫标记的孤立的豚鼠肝间隙连接斑块与Cx 32或Cx 26抗血清产生完整和致密的抗体装饰的斑块的细胞质表面。我们在检查的任何斑块中均未观察到标记的任何局灶性或斑片状簇。双标记免疫电镜证实，Cx 32和Cx 26共定位在同一间隙连接斑块。这些结果表明，在肝细胞表达的Cx 32和Cx 26，这两种类型的间隙连接蛋白是不分离，但混杂随机在同一plake.Cytoplasmic表面结构的牛透镜纤维间隙连接：牛透镜纤维的快速冷冻深蚀刻复制方法研究显示，在原位细胞质表面上的颗粒亚结构。在分离的膜中，薄切片电子显微镜显示16-17 nm的五层连接包括在该标本中。深蚀刻复制品显示，间隙连接斑块也有颗粒在其细胞质表面上，清楚地区别于周围的无颗粒的非间隙连接表面。用蛋白内切酶glu-C处理分离的透镜膜，在SDS-PAGE中诱导MP 70裂解为MP38，并丧失其对抗MP 70单克隆抗体的抗原性，而颗粒图案仍然持续存在于间隙连接斑块的细胞质表面，这表明在透镜纤维间隙连接结构中存在除MP 70以外的其他蛋白。

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.