Manjunath C K, Goings G E, Page E
Biochem J. 1982 Jul 1;205(1):189-94. doi: 10.1042/bj2050189.
We have modified a method for isolating gap-junctional membrane from mouse hearts [Kensler & Goodenough (1980) J. Cell Biol. 86, 755-764] to isolate gap junctions of comparable purity from rabbit hearts more rapidly, with better yield, and without resort to non-ionic detergents. Purification was monitored by electron microscopy of thin-sectioned membrane pellets and by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Gap junctions were obtained as vesicles whose mean surface area approximated that of junctions in intact myocardial cells. About 10-20% of the vesicles were ferritin-impermeable. Approx. 125 micrograms of membrane protein was obtained per 8 g of rabbit heart. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of purified gap junctions showed five major protein bands of mol.wts. 46 000, 44 000, 33 000, 30 000 and 28 500 that co-purified with the junctions. This protein composition was nearly identical with that published for gap junctions of mouse hearts, and differed markedly from the protein composition of gap junctions from non-excitable cells (lens and liver). The constancy of junctional protein composition between hearts of two different species and its non-identity with that from liver and lens suggest that, although gap-junctional structure in mammalian tissues seems to be remarkably similar by electron-microscopic techniques, junctional-channel protein composition actually varies from tissue to tissue and may be adapted to the permeability requirements of the tissue.
我们改进了从小鼠心脏中分离间隙连接膜的方法[肯斯勒和古德诺夫（1980年）《细胞生物学杂志》86卷，755 - 764页]，以便能更快速地从兔心脏中分离出纯度相当的间隙连接，产量更高，且无需使用非离子去污剂。通过对薄切片膜沉淀进行电子显微镜观察以及十二烷基硫酸钠/聚丙烯酰胺凝胶电泳来监测纯化过程。间隙连接以囊泡形式获得，其平均表面积接近完整心肌细胞中连接的表面积。约10 - 20%的囊泡对铁蛋白不可渗透。每8克兔心脏可获得约125微克膜蛋白。纯化后的间隙连接的十二烷基硫酸钠/聚丙烯酰胺凝胶电泳显示出五条主要蛋白带，分子量分别为46000、44000、33000、30000和28500，它们与连接一起共纯化。这种蛋白质组成与已发表的小鼠心脏间隙连接的蛋白质组成几乎相同，与非兴奋性细胞（晶状体和肝脏）的间隙连接的蛋白质组成明显不同。两种不同物种心脏之间连接蛋白组成的稳定性及其与肝脏和晶状体连接蛋白组成的不同表明，尽管通过电子显微镜技术哺乳动物组织中的间隙连接结构似乎非常相似，但连接通道蛋白组成实际上因组织而异，并且可能适应组织的通透性需求。
