Molecular structural studies of gap junction protein compositions.

利用特异性兔抗体，在单个连接子通道水平、组织分布和细胞类型特异性上，检测了连接蛋白（Cx）家族的缝隙连接蛋白.通过温和去污剂提取从大鼠和豚鼠肝脏分离的单个间隙连接通道（connexon）颗粒在负染色和低角度旋转阴影电子显微镜下显示具有中央凹陷的甜甜圈状单位颗粒。通过高效液相色谱法进一步纯化，然后在抗Cx 26或抗Cx 32特异性抗体柱上进行亲和色谱，得到单一的蛋白质印迹模式，没有明显的其他连接蛋白的共沉淀。这一结果可能表明单个连接子六聚体仅由Cx 26或Cx 32组成.与含有Cx 32和Cx 26的唾液腺相反，分泌脂质的哈氏腺腺泡细胞中仅显示Cx 26免疫荧光。肌上皮细胞显示Cx43。在乳腺中，腺泡细胞中的Cx 26和Cx 32以及肌上皮细胞中的Cx43在RNA转录、蛋白质产生和磷酸化状态方面显示出在妊娠期、分娩期、哺乳期和断奶期的动态变化，显示活动-这些结果表明，不同的间隙连接蛋白的差异表达，与细胞类型特异性功能和活性密切相关。

Gap junction proteins of connexin (Cx) family were examined by using specific rabbit antibodies raised against type-specific peptide sequence domains at the single connexon channel level and on the tissue distiribution and cell type secificity.1. Single gap junction channel (connexon) particles isolated from rat and guinea pig liver by mild detergent extraction showed doughnut-shaped unit particles with central depressions in negative staining and low angle rotary shadowing electron microscopy. Further purification by high performance liquid chromatography followed by affinity chromatogram on either anti Cx26 of anti-Cx32 specific antibody column yielded single western blot pattern without appreciable cosedimentations of other connexins. This result may indicate that single connexon hexamers are composed of either Cx26 of Cx32 only.2. In contrast to the salivary glands which contained both Cx32 and Cx26, lipid secreting Harderian glands showed only Cx26 immunofluorescence in the acinar cells. Myoepithelial cells showed Cx43. In mammary glands, Cx26 and Cx32 in acinar cells and Cx43 in myoepithelial cells revealed dynamic changes of expression during pregnancy, parturition, lactation and weaning period at RNA transcription, protein production, and phosphorylation states, indicating the activity-related expression of gap junction proteins thus consituting functionally distinct cell groups.These results indicate that differential expressions of various gap junction proteins are closely correlated with cell type specific functions and activities.