Goodenough D A
J Cell Biol. 1976 Feb;68(2):220-31. doi: 10.1083/jcb.68.2.220.
A method is described that uses trypsin digestion combined with collagenase-hyaluronidase which produces a population of gap junction vesicles. The hexagonal lattice of subunits ("connexons") comprising the gapjunctions appears unaltered by various structural criteria and by buoyant density measurements. The gap junction vesciles are closed by either a single or a double profile of nonjunctional "membrane," which presents a smooth, particle-free fracture face. Horseradish peroxidase and cytochrome c studies have revealed that about 20% of the gap junction vesicles are impermeable to proteins 12,000 daltons or larger. The increased purity of the trypsinized junction preparation suggests that one of the disulfide reduction products of the gap-junction principal protein may be a nonjunctional contaminating peptide. The gap junction appears to be composed of a single 18,000-dalton protein, connexin, which may be reduced to a single 9,000-dalton peak. The number of peptides in this reduced peak are still unknown.
本文描述了一种方法，该方法使用胰蛋白酶消化结合胶原酶 - 透明质酸酶来产生一群间隙连接囊泡。组成间隙连接的亚基（“连接子”）的六边形晶格，根据各种结构标准和浮力密度测量，看起来未发生改变。间隙连接囊泡由单层或双层非连接“膜”封闭，其呈现出光滑、无颗粒的断裂面。辣根过氧化物酶和细胞色素c研究表明，约20%的间隙连接囊泡对12,000道尔顿或更大的蛋白质是不可渗透的。胰蛋白酶处理后的连接体制剂纯度提高，这表明间隙连接主要蛋白质的二硫键还原产物之一可能是一种非连接污染肽。间隙连接似乎由单一的18,000道尔顿蛋白质连接蛋白组成，该蛋白可能被还原为单一的9,000道尔顿峰。这个还原峰中的肽数量仍然未知。
