Zimmer D B, Green C R, Evans W H, Gilula N B
J Biol Chem. 1987 Jun 5;262(16):7751-63.
The topological organization of the major rat liver gap junction protein has been examined in intact gap junctions and gap junction-derived single membrane structures. Two methods, low pH and urea at alkaline pH, were used to "transform" or "split" double membrane gap junctions into single membrane structures. Low pH treatment "transforms" rat liver gap junctions into small single membrane vesicles which have an altered sodium dodecyl sulfate-polyacrylamide gel electrophoresis profile after digestion with L-1-to-sylamido-2-phenylethylchloromethyl ketone-trypsin. Alkaline pH treatment in the presence of 8 M urea can split isolated rat liver gap junctions into single membrane sheets which have no detectable structural alteration or altered sodium dodecyl sulfate-polyacrylamide gel electrophoresis profile after proteolytic digestion, suggesting that these single membrane sheets may be useful for topological studies of the gap junction protein. Proteolytic digestion studies have been used to localize the carboxyl terminus of the molecule on the cytoplasmic surface of the intact gap junction. However, the amino terminus does not appear to be accessible to proteases or to interaction with an antibody that is specific for the amino-terminal region of the molecule in intact or split gap junctions. Binding of antibodies, that block junctional channel conductance, can be eliminated by proteolytic digestion of intact gap junctions, suggesting that all antigenic sites for these antibodies are located on the cytoplasmic surface of the intact gap junction. In addition, calmodulin gel overlays indicate that at least two calmodulin binding sites exist on the cytoplasmic surface of the junctional protein. The information generated from these studies has been used to develop a low resolution two-dimensional model for the organization of the major rat liver gap junctional protein in the junctional membrane.
已在完整的间隙连接和间隙连接衍生的单膜结构中研究了大鼠肝脏主要间隙连接蛋白的拓扑组织。使用低pH值和碱性pH值的尿素这两种方法将双膜间隙连接“转化”或“拆分”为单膜结构。低pH处理将大鼠肝脏间隙连接“转化”为小的单膜囊泡，在用L-1-对甲苯磺酰胺基-2-苯乙基氯甲基酮-胰蛋白酶消化后，其十二烷基硫酸钠-聚丙烯酰胺凝胶电泳图谱发生改变。在8M尿素存在下的碱性pH处理可将分离的大鼠肝脏间隙连接拆分为单膜片，这些单膜片在蛋白水解消化后没有可检测到的结构改变或十二烷基硫酸钠-聚丙烯酰胺凝胶电泳图谱改变，这表明这些单膜片可能对间隙连接蛋白的拓扑研究有用。蛋白水解消化研究已用于将该分子的羧基末端定位在完整间隙连接的细胞质表面上。然而，在完整或拆分的间隙连接中，氨基末端似乎无法被蛋白酶作用或与针对该分子氨基末端区域的特异性抗体相互作用。阻断连接通道电导的抗体的结合可通过对完整间隙连接进行蛋白水解消化来消除，这表明这些抗体的所有抗原位点都位于完整间隙连接的细胞质表面上。此外，钙调蛋白凝胶覆盖实验表明，连接蛋白的细胞质表面上至少存在两个钙调蛋白结合位点。这些研究产生的信息已用于建立大鼠肝脏主要间隙连接蛋白在连接膜中组织的低分辨率二维模型。
